Microfluidic viscometer

ABSTRACT

Microfluidic devices, systems, and methods measure viscosity, flow times, and/or pressures. other flow characteristics within the channels, and the measured flow characteristics can be used to generate a desired flow. Multi-reservoir pressure modulator and pressure controller systems, electrokinetic systems and/or other fluid transport mechanisms can generate the flow, controllably mix fluids, and the like.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 10/008,604, filed Nov. 9, 2001, which is a continuation-in-partof U.S. application Ser. No. 09/792,435 filed on Feb. 23, 2001. Thisapplication claims priority to and benefit of these prior applications.This application also claims the benefit of and priority to U.S.Provisional Patent Application No. 60/216,793 filed on Jul. 7, 2000, andU.S. Provisional Patent Application No. 60/184,390 filed Feb. 23, 2000.The full disclosures of each of these prior applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention is generally related to analytical toolsfor the biological and chemical sciences, and in a particularembodiment, provides microfluidic devices, systems, and methods fordetermining the viscosity of fluids within microfluidic channels of amicrofluidic network, optionally without adding dye (or other agents)which can alter the properties of the fluids.

[0003] Microfluidic systems are now in use for the acquisition ofchemical and biological information. These microfluidic systems areoften fabricated using techniques commonly associated with thesemiconductor electronics industry, such as photolithography, wetchemical etching, and the like. As used herein, “microfluidic” means asystem or device having channels and chambers which are at the micron orsubmicron scale, e.g., having at least one cross-sectional dimension ina range from about 0.1 μm to about 500 μm.

[0004] Applications for microfluidic systems are myriad. Microfluidicsystems have been proposed for capillary electrophoresis, liquidchromatography, flow injection analysis, chemical reaction andsynthesis, and many other uses. Microfluidic systems also have wideranging applications in rapidly assaying compounds for their effects onvarious chemical, and, preferably, biochemical systems. Theseinteractions include the full range of catabolic and anabolic reactionswhich occur in living systems, including enzymatic, binding, signaling,and other reactions.

[0005] A variety of methods have been described to effect the transportof fluids between a pair of reservoirs within a microfluidic system ordevice. Incorporation of mechanical micro pumps and valves within amicrofluidic device has been described to move the fluids within amicrofluidic channel. The use of acoustic energy to move fluid sampleswithin a device by the effects of acoustic streaming has been proposed,along with the use of external pumps to directly force liquids throughmicrofluidic channels.

[0006] The capabilities and use of microfluidic systems advancedsignificantly with the advent of electrokinetics: the use of electricalfields (and the resulting electrokinetic forces) to induce flow of fluidmaterials through the channels of a microfluidic system. Electrokineticforces have the advantages of direct control, fast response, andsimplicity, and allow fluid materials to be selectively moved through acomplex network of channels so as to provide a wide variety of chemicaland biochemical analyses. An exemplary electrokinetic system providingvariable control of electro-osmotic and/or electrophoretic forces withina fluid-containing structure is described in U.S. Pat. No. 5,965,001,the full disclosure of which is incorporated herein by reference.

[0007] Despite the above-described advancements in the field ofmicrofluidics, as with all successes, still further improvements aredesirable. For example, while electrokinetic material transport systemsprovide many benefits in the micro-scale movement, mixing, andaliquoting of fluids, the application of electrical fields can havedetrimental effects in some instances. In the case of charged reagents,electrical fields can cause electrophoretic biasing of material volumes,e.g., highly charged materials moving to the front or back of a fluidvolume. Where transporting cellular material is desired, elevatedelectrical fields can, in some cases, result in a perforation orelectroporation of the cells, which can effect their ultimate use in thesystem.

[0008] To mitigate the difficulties of electrokinetic systems,simplified transport systems for time domain multiplexing of reagentshas been described in WO 00/45172 (assigned to the assignee of thepresent invention), the full disclosure of which is incorporated hereinby reference. Still further alternative fluid transport mechanisms andcontrol methodologies to enhance the flexibility and capabilities ofknown microfluidic systems, including multiple modulated pressure-driventechniques, have been described in International Application No.PCT/US01/05960, the full disclosure of which is also incorporated hereinby reference.

[0009] Regardless of the mechanism used to effect movement of fluid andother materials within a microfluidic channel network, accuracy andrepeatability of microfluidic flows can be problematic. Quality controlcan be challenging in light of variability of the fluids making up theseflows, and accurate control over microfluidic flows in applications suchas high throughput screening would benefit significantly from stable andreliable assays. It would also be beneficial to determine additionalcharacteristics of the fluids flowing within the microfluidic channelsof a microfluidic network.

[0010] In light of the above, it would be advantageous to provideimproved microfluidic devices, systems, and methods. It would bedesirable if these improved techniques allowed better control over theflows within a microfluidic network, and/or increased the informationprovided by the microfluidic systems regarding one or more of thecharacteristics of the fluids flowing within a microfluidic channel ofthe network. It would be particularly beneficial if these enhancedtechniques provided real-time and/or quality control feedback on theactual flows, ideally without relying on significantly increased systemcomplexity or cost.

SUMMARY OF THE INVENTION

[0011] The present invention generally provides improved microfluidicdevices, systems, and methods. The devices and systems of the inventiongenerally allow the characteristics of a fluid within a microfluidicsystem to be determined, often using high-throughput techniques. In manyembodiments, the devices and systems will determine the viscosity of oneor more sample fluids within a microfluidic channel network of amicrofluidic body. The microfluidic networks generally include at leastone flow-resisting channel segment, and viscosity can be determined byflowing the sample fluid through the channel segment, often withoutaltering the sample viscosity, by adding any detectable marker (such asfluorescent dyes or the like) to the fluid before it flows through thechannel segment. These techniques can also allow the use of dyes whichare not normally compatible with a particular sample fluid, for example,dyes which are not soluble or the like. The viscosity can be determinedby mixing the sample fluid with a detectable marker at an intersectiondownstream of the flow-resisting channel segment, with the mixingcharacteristics at the intersection indicating the pressure drop alongthe channel segment (and hence the viscosity of the sample fluid).Viscosities can be determined by comparing the flow characteristics ofthe sample fluid with a reference fluid having a known viscosity. Thesensing range can be enhanced using a plurality of flow-resistingchannel segments and/or detectable fluid channel intersections.

[0012] In a first aspect, the invention provides a microfluidicviscometer system comprising a microfluidic channel network including afirst flow-resisting channel segment. A sensor coupled to the firstsegment of the network determines a viscosity of a sample fluid therein.

[0013] In many embodiments, a body having channel walls defines thenetwork. The network can include a plurality of channels with one ormore intersections therebetween. A flow generator (e.g., a pressure orvoltage source) coupled to the network can induce a flow of the samplefluid within the first segment. A first intersection can be incommunication with the first segment, with the sensor coupled to thenetwork at a sensor location disposed downstream of the first segment.This allows the sensor to sense a change in the flow which propagatesfrom the first intersection to the sensor location so as to determinethe viscosity of the sample fluid.

[0014] In some embodiments, the change in flow comprises a pulse of adetectable fluid introduced at the first intersection, which isoptionally upstream of the first segment. The system can then determinethe viscosity of the sample fluid using steady state propagation of theflow (which includes the detectable fluid pulse) from the intersectionthrough the first segment and to the sensor location. In suchembodiments, it is possible that the presence of the detectable fluidpulse can, to some extent, alter the characteristics (including theviscosity) of the sample fluid flowing through the first segment.Related embodiments can make use of a step-function change in flow of adetectable fluid.

[0015] In alternative embodiments, the first segment is optionallydisposed upstream of the first intersection. The flow defines a ratiobetween a quantity of a sample fluid in the flow and a quantity of adetectable fluid in the flow, the detectable fluid being detectable bythe sensor and traversing a second flow-resisting channel segmentbetween a detectable fluid source and the intersection. By monitoringthe changes in the mixing ratio, typically by monitoring the strength ofa detectable signal provided from the mixed flow, a processor coupled tothe sensor can determine the viscosity of the sample fluid.Advantageously, the viscosity sensing techniques of the presentinvention are particularly well-suited for sequential viscositymeasurements of a plurality of sample fluids, particularly when thefluids are transferred along a fluid introduction channel in the form ofa capillary extending or protruding from the microfluidic body.

[0016] The microfluidic systems of the invention can include, e.g., achannel split to a pair of channels having different hydrodynamicresistance, wherein a fluid can flow for detection of a transit timedifferential related to the viscosity of the fluid. A first segment,with an upstream end, a downstream end, can be intersected at each endby a second flow-resisting segment with a different hydrodynamicresistance. The system can include, e.g., an instruction set to comparetransit times of a fluid through the first segment and the secondsegment for determination of a fluid viscosity.

[0017] The microfluidic systems of the invention can optionally beprovided with a side channel pressure sensor to measure pressures at anypoint along a channel. For example, a side channel segment can runbetween a side channel well and an intersection with a channelintersection where a pressure is to be measured. A detector can becoupled to the side channel segment to detect flow (or zero-flow). Apressure sensor can be coupled, e.g., to the side channel well tomeasure a side channel well pressure and the pressure at the channelintersection, e.g., when zero-flow conditions have been established inthe side channel segment.

[0018] In another embodiment of the systems of the invention fordetermination of fluid viscosity, e.g., a sample fluid channel andreference fluid channel can be connected by a common diluent fluidchannel so that the relative viscosity of a sample and reference can becompared. A sample fluid containing flow-resisting channel can be, e.g.,in fluid contact with a reference fluid containing channel through adiluent channel. The a diluent fluid can include, e.g., a detectablemarker detectable by sensors coupled to the sample channel and/or thereference channel. The system of this embodiment can provide, e.g.,pressure, timing and/or detectable marker quantity output values thatcan be used to calculate sample fluid viscosity.

[0019] A further embodiment of microfluidic viscometer system of theinvention directs reference and sample fluids to flow together at aT-intersection resulting in a non-mixing interface with a location thatreflects the relative viscosities of the two fluids. The systemincludes, e.g., a sample fluid channel, a reference fluid channel, and adetector channel that come together in a T-intersection. A detector iscoupled to the detector channel to detect, e.g., the location of the afluid interface within the detector channel. Fluid channels can have,e.g., pressure detectors (such as readout from a multiport pressuremanifold system) to report pressure levels, e.g., required to center theinterface in the detector channel. Such values can be input to formulasfor calculations of fluid viscosity.

[0020] In one aspect of the systems of the invention, viscoelasticitycan be measured, e.g., by inducing an oscillating flow into a samplechannel and detecting resultant marker concentration oscillations ofsample co-flowing with a reference fluid in a detection channel. Amicrofluidic viscoelasticity measurement system can include, e.g., asample fluid channel in fluid contact with a flow generator forinduction of an oscillating sample fluid flow, a reference fluid channelin fluid contact with the sample fluid channel at an intersection, adetector channel in fluid contact with the sample channel and thereference channel at the intersection, and a detector coupled to thedetector channel. The detector can be configured, e.g., to detectoscillations in the amount of detectable marker a fluid. An instructionset can be configured, e.g., to determine a phase shift between a fluidoscillation and a detectable marker oscillation for determination of afluid viscoelasticity.

[0021] In a method aspect, the invention comprises determining aviscosity of a sample fluid. The method comprises altering a flow of aflow-restricting microfluidic channel segment. The viscosity of thesample fluid can be determined, e.g., by monitoring the altered flow.

[0022] In many embodiments, a first flow of a reference fluid throughthe flow-resisting channel will be monitored, the reference fluid havinga known viscosity. A second flow through the flow-resisting channel willalso be monitored, the second flow comprising the sample fluid. Theviscosity of the sample fluid is optionally determined at least in partby comparing the first and second flows, with calculations based in parton the known viscosity of the reference fluid. The first and secondflows can be monitored by a sensor disposed downstream of theflow-resisting channel with an intersection disposed between the flowresisting channel and the sensor. The flows can be monitored, e.g., bysensing a ratio of the sample fluid to a detectable fluid.Advantageously, a plurality of sample fluids can be sequentiallytransferred to the flow-resisting channel segment, allowing theviscosities of the samples to be determined in a high-throughput manner.In some embodiments, fluids are dispensed and/or mixed within amicrofluidic network, for example, allowing viscosities of fluidmixtures to be determined as a function of their composition.

[0023] In another aspect, the invention comprises a microfluidic channelnetwork including a first flow-resisting channel segment. A sensor iscoupled to the network for sensing flows through the first segment. Aprocessor is coupled to the sensor. The processor derives a viscosity ofa sample fluid by comparing first and second flows through the firstsegment.

[0024] The system can further include a reference fluid disposed withinthe network. The first flow can comprise the reference fluid, and thesecond flow can comprise the sample fluid. In many embodiments, thesecond flow within the first segment is substantially composed of thesample fluid. The processor can calculate the viscosity of the samplefluid based at least in part on a viscosity of the reference fluid.

[0025] In many embodiments, a second flow-resisting channel segment iscoupled to the first segment at a first intersection. A first detectablefluid can be disposed within the second segment. The first intersectioncan be downstream of the first segment, and the sensor can monitor theflow through the first segment by sensing a quantity of the firstdetectable fluid added to the flow at the first intersection. Stillfurther additional flow resisting channel segments can be coupled to thefirst segment by additional intersections. The intersections canoptionally be separated by associated flow-resisting channel segments,and the sensor can monitor the flow by sensing a quantity of the firstdetectable fluid added to the flow at the intersection. Alternatively,one or more additional flow-resisting channel segments can be coupled tothe first segment, with the sensor monitoring the flow through the firstsegment by sensing a quantity of a second detectable fluid added to theflow through the third segment. In such embodiments, the second andthird segments can have differing resistances to flows therein. Thefirst and second detectable fluids can be independently detectable bythe sensor, for example, comprising dyes having differing color (orother detectable markers) signatures.

[0026] The first segment can comprise a channel having a locallyenhanced resistance to flows therein. For example, the channel regioncan have a reduced cross-sectional dimension, such as a reduced depth, areduced width, or the like. Alternatively, a flow occluding structurecan be disposed within the channel.

[0027] The first flow optionally comprises a reference fluid having aknown viscosity, and the second flow optionally comprises a combinationof the sample fluid and a detectable fluid. This combination can definea ratio, with the processor identifying the ratio from a signal producedby the sensor. For example, the sensor can sense a light signalgenerated by a fluorescent dye of the detectable fluid, with a relativestrength of the fluorescence indicating the ratio.

[0028] In many embodiments, the processor derives the viscosity of thesample fluid by determining a rate of change of a signal generated bythe sensor. In some embodiments, the processor derives the viscosity ofthe sample fluid by determining a magnitude of a change of a signalgenerated by the sensor. The processor can determine the sampleviscosity throughout a range of at least about two orders of magnitudeof cp (centipoise) units, preferably through a range of at least threeorders of magnitude of cp units. The processor can determine the sampleviscosity throughout at least a range from about 1 cp to 100 cp,preferably from about 1 cp to 1000 cp, and optionally from 0.1 cp to1000 cp. By, for example, simply altering driving pressures, largerviscosities are sensed, i.e., 1,000 cp-100,000 cp. Hence, at leastviscosities throughout ranges of at least 2 or 3 orders of magnitude canbe sensed using the systems of this embodiment.

[0029] A microfluidic system optionally includes a sample fluid sourcewhich includes a plurality of sample fluids and a sample fluidintroduction channel. The sample fluids can then be sequentiallytransferable along the fluid introduction channel to the flow resistingchannel so as to sequentially determine viscosities of the sample fluid.The sample introduction channel optionally comprises a capillaryextending from the microfluidic body, with the capillary beingextendable sequentially (or with multiple capillaries in parallel) intothe sample fluids. Generally, the capillary will have significantly lessresistance to flow than the first segment.

[0030] In yet another embodiment, the invention provides a microfluidicsystem comprising a microfluidic body having a network of channels. Aflow generator induces a flow within the network, and a sensor transmitsa signal indicating a time of the flow. A processor effects feedbackcontrol of the flow in response to the time signal. Optionally, theprocessor can determine a viscosity for use in the feedback controlloop.

[0031] Yet another embodiment of the invention provides a microfluidicsystem comprising first and second immisciable fluids. A microfluidicbody having a network of channels combines the fluids therein, and asensor is coupled to the network so as to define a viscometer. Theviscometer measures interfacial properties of the combined fluid.

[0032] A method for determining the viscosity of a sample fluid caninclude, e.g., flowing the sample fluid through two or more microfluidicchannel segments of different hydrodynamic resistance, and monitoringthe difference in transit times of sample fluid in the channels so thatthe viscosity of the sample fluid can be determined based in part on thedifference in transit times. The method can optionally include, e.g.,comparing the difference in transit times of the sample fluid to thedifference in transit times for a reference fluid of known viscosity forviscosity determinations based in part on the known viscosity of thereference fluid.

[0033] An alternative method of the invention for determining theviscosity of a sample fluid can be based, e.g., on parameters affectingthe location of a non-mixed sample fluid/reference fluid interfaceflowing in a detection channel. The method can include, e.g., flowingthe sample fluid in a first microfluidic channel, flowing the referencefluid in a second microfluidic channel converging with the first channelat a T-intersection, to form a non-mixed interface flowing from theT-intersection into a third micro channel. The location of the interfacein the third channel can be adjusted by manipulating the forces inducingflow of the fluids. The viscosity of the sample fluid can be determinedbased in part, e.g., on the difference in pressures between the firstand second channels, and on the known viscosity of the reference fluid.

[0034] In other embodiments, methods of the invention can includedetermining the viscosity, e.g., of a sample fluid flowing in onemicrofluidic channel relative to a reference standard flowing in anothermicrofluidic channel, where both micro channels are in fluid contactwith a diluent channel containing a detectable marker. For example, themethod can comprise flowing the reference fluid of known viscosity in areference channel intersected by the diluent channel, flowing the samplefluid in the sample channel intersected by the diluent channel, flowingthe diluent fluid into the reference and/or sample channels, anddetecting the diluent fluid in the sample and/or reference channels. Theviscosity of the sample fluid can be determined, e.g., based on outputvalues, such as the relative amounts of detectable marker in the sampleand reference streams, the relative time required for a detectablemarker pulse to travel with the sample and reference fluids to adetector, and/or the relative pressures required to induce a detectablemarker pulse to simultaneously traverse the reference and samplechannels. In one embodiment, the viscosity of the sample fluid can bedetermined based on the difference in amounts of detectable markerdetected in the sample channel and the reference channel givenequivalent flow inducing pressures. In another embodiment, a pulse ofdetectable marker can be injected from the diluent channel into thesample and reference channels, the travel times of the pulse in the twochannels measured, and the viscosity of the sample fluid determinedbased on the travel time difference for the pulses in the two channels.In still another embodiment, the methods can include monitoringpressures inducing flow in the sample and reference channels, injectinga pulse of detectable marker from the diluent channel into the channels,and determining the viscosity of the sample fluid based in part on thedifference in pressures used to induce flow in the two channels.

[0035] The present invention provides methods for determining, e.g., thekinematic viscosity and/or the mass-percent composition of a fluidadmixed in a microfluidic system with one or more other fluids. Themethod can include, e.g., flowing one or more fluids into a microfluidicchannel, setting a side channel pressure on a side channel intersectingthe microfluidic channel at a first location to provide a zero-flowcondition in the side channel to determine the intersection pressure,and determining the pressure difference between the intersectionpressure and the microfluidic channel pressure at a second location,thereby providing parameters for determination of the viscosity ormass-percent composition of the fluid mixture. Setting the side channelpressure can comprise detecting a marker in a side channel fluid, andadjusting the side channel pressure to provide unchanging detection ofthe marker to provide a zero-flow condition wherein a pressure measuredat the side well is an indirct measure of the pressure at theintersection. The detector of a zero-flow condition on the side channelcan be configured to measure conductivity, fluorescence, lightabsorption, refraction, and/or the like.

[0036] The present invention provides methods for determining theviscoelasticity of a sample fluid. The method can include, e.g., flowinga reference fluid in a reference microfluidic channel which intersects asample microfluidic channel to flow into a common detector channel. Anoscillating flow of sample fluid can be, e.g., induced in the samplechannel by a flow generator to flow into the detector channel. Thesample fluid and/or reference fluid can contain, e.g., a detectablemarker. Oscillations in the amount of detectable marker can result,e.g., in the detector channel, which can be compared to the flowoscillations induced by the flow generator in the sample fluid todetermine a phase shift between the induced and detected oscillations.The viscoelasticity of the sample fluid can thereby be calculated basedin part on the phase shift.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1 schematically illustrates a microfluidic system having amulti-reservoir pressure modulation system according to the principlesof the present invention.

[0038]FIG. 2 is a plan view of a representative microfluidic devicehaving microfluidic channels with enhanced fluid flow resistance for usein the microfluidic system of FIG. 1.

[0039]FIGS. 3A and 3B are perspective views of a pressure manifold forreleasably sealing reservoirs of the microfluidic device of channel 2 influid communication with the pressure modulators of the system of FIG.1.

[0040]FIG. 4 schematically illustrates a control system forindependently varying reservoir pressures in the microfluidic system ofFIG. 1.

[0041] FIGS. 5A-C schematically illustrate a method and computer programfor determining pressures to provide a desired flow within a channel ofthe microfluidic network in the microfluidic device of FIG. 2.

[0042]FIG. 6 schematically illustrates a microfluidic system having botha multi-reservoir pressure modulation system and an electrokinetic fluidtransportation and control system according to the principles of thepresent invention.

[0043]FIGS. 7A and 7B illustrate well-pair dilution in whichconcentration variations are produced by selectively varying therelative flow rates from two reservoirs connected at an intersection.

[0044] FIGS. 7C-E graphically illustrate measured dilution vs. set orintended dilution for a multi-reservoir pressure controlled well-pairdilution.

[0045]FIGS. 8 and 8A-8D graphically illustrate an enzyme assay using amulti-reservoir pressure controlled microfluidic system, and morespecifically: FIG. 8 illustrates the reaction, FIG. 8A is a titrationcurve for different substrate concentrations, FIG. 8B is a plot of thecorrected signal verses substrate concentration, FIG. 8C is a plot fordetermination of the Michaelis constant, and FIG. 8D is a substratetitration plot.

[0046] FIGS. 9A-C illustrate a microfluidic Protein Kinase A (PKA)reaction assay with variations in concentration achieved usinghydrodynamic pressure modulation.

[0047]FIGS. 10A and 10B illustrate a mobility shift assay microfluidicnetwork and assay test results at different concentrations.

[0048]FIGS. 11A and 11B are a perspective and plane view, respectively,of an exemplary hydrodynamic and electrokinetic interface structure forcoupling to a microfluidic body.

[0049]FIG. 12 schematically illustrates an exemplary microfluidicviscometer.

[0050]FIGS. 13A, 13B, and 13C schematically illustrate microfluidicnetworks and method for imposing detectable signals on a microfluidicflow for measurement of flow characteristics which can be used tocalculate pressures to affect a desired flow, for viscometry, and thelike.

[0051]FIGS. 14A and 14B graphically illustrate flow characteristicsignals which can be used to determine effective viscosity.

[0052]FIG. 15 is a perspective view of a microfluidic chip having aplurality of capillaries for spontaneous injection of fluids into themicrofluidic network, typically by introducing the capillaries intofluid sources to bring the fluid into the chip in a controlled fashion.

[0053]FIG. 16 is a top view of a simple microfluidic chip having asingle capillary for spontaneous injection.

[0054] FIGS. 16A-16C graphically illustrate methods for monitoringprogress of perturbations induced by spontaneous injection of fluids,for use in determining characteristics of a flow and/or microfluidicnetwork.

[0055]FIGS. 17A and 17B are perspective and plan view of fluorogenicmulti-capillary chips.

[0056]FIGS. 18A and 18B are perspective and plan view of amobility-shift capillary chip.

[0057]FIG. 19 graphically illustrates the detection of a perturbationgenerated at an intersection of microfluidic channels by spontaneousinjection.

[0058]FIG. 20 schematically illustrates a microfluidic network for usein a microfluidic system for determining a viscosity of a sample fluidwithout adding dyes, or the like, so as to distort the viscositymeasurement, and/or to allow the use of dyes that are not compatiblewith the sample.

[0059]FIGS. 21A and 21B schematically illustrate signals provided by thesensor of the microfluidic network of FIG. 20, allowing determination ofa viscosity of a sample fluid relative to a known viscosity of areference fluid.

[0060]FIG. 22 schematically illustrates a microfluidic network in whicha locally enhanced flow resistance region is used as a portion of aviscometer.

[0061]FIGS. 23A and 23B schematically illustrate signals provided by thesensor associated with the network of FIG. 22.

[0062]FIG. 24 schematically illustrates yet another alternativemicrofluidic network for use in a microfluidic viscometer system inwhich a series of sequential channel segments are coupled to a series ofdetectable fluid sources at a series of intersections so as to determineviscosities throughout a wide range.

[0063]FIG. 25 schematically illustrates a microfluidic viscometrynetwork in which independently detectable fluids are coupled to a samplefluid flow resisting channel by detectable fluid channel segments havingdiffering flow resistance so as to identify sample fluid viscositiesthroughout a wide range.

[0064]FIG. 26 schematically illustrates a system for sequentiallyintroducing sample fluids into a microfluidic body.

[0065] FIGS. 27-33 graphically illustrate measurements and computermodel data for determining viscosity and calibrating viscositymeasurements.

[0066]FIGS. 34A to 34F schematically illustrate apulsed-injection/perturbation method for determination of viscosity.

[0067]FIG. 35 schematically illustrates a system for determining fluidviscosity using a method of pressure adjustments to a fluid interfacelocation.

[0068]FIG. 36 graphically illustrates calibration curves for interfacelocation fluid viscosity measurement systems having different detectionchannel aspect ratios.

[0069] FIGS. 37A-37B schematically illustrate systems for determiningmass-percent composition and kinematic viscosity of fluids.

[0070]FIG. 38 schematically illustrates a system with paired channelgeometry and a common detectable marker diluent fluid source fordetermination of fluid viscosities.

[0071]FIG. 39 schematically illustrates a system for determining fluidviscoelasticity.

[0072]FIG. 40 schematically illustrates a surfactant micellar phasechange that can affect fluid viscoelasticity.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0073] The present invention will provide improved microfluidic devices,systems, and methods. The systems of the present invention will oftendetermine a viscosity (or other characteristics) of a sample fluid bymaking use of the resistance to flow present within the small channelsof a microfluidic network. Viscosities can be determined without, forexample, adding fluorescent dyes or other detectable substances in amanner which could alter or distort the indicated viscosity. Toward thatend, the fluid sample can flow through a flow resisting channel whilesample fluid is substantially free of a substance which is detectable tothe sensor.

[0074] As used herein, a sample is “substantially free” of a detectablesubstance if an associated viscosity sensor does not receive asufficient signal from the detectable substance so as to identify thepresence or speed of the detectable substance. It should be noted thatsuch detectable substances can be, and often are, added to the samplefluid flow downstream of the flow-resisting channel segment.Nonetheless, as it flows through the flow-resisting channel segment (andhence as its viscosity is measured) the fluid can still be substantiallyfree of the detectable substance. As used herein, a sensor having adetection region in fluid communication with a microfluidic network (orsome component thereof) is encompassed within the term “a sensor coupledto” the microfluidic network (or component thereof).

[0075] Preferably, viscosities of fluid samples of no more than about 30μl can be determined, ideally providing viscosities with fluid samplesvolumes of less than 5 μl. In fact, only a few (about 3 or more)nanoliters of a sample fluid can be brought into the network, so thatviscosities of this quantity of fluid or more can be determined in someembodiments, with many embodiments able to measure as little as 10nanoliters, typically allowing samples of 100 nanoliters or more to bemeasured. When used to determine viscosities, flow of a sample fluid canbe generated by applying positive-gauge pressures, by applying vacuum ornegative-gauge pressures, by electrokinetic microfluidic techniques, orthe like. When a fluid sample flows through a flow-resisting channel,the pressure loss can be determined by providing an intersectiondownstream of the flow-resisting channel segment. Specifically, bycoupling a detectable fluid source to the intersection via a second flowresisting channel segment, and (for example) drawing a vacuum downstreamof the intersection, the sample and detectable fluid is drawn and mixedin a ratio which depends in part on the viscosities of the sample anddetectable fluid. Determination of the viscosity of a particular samplefluid can be simplified by flowing a reference fluid through theflow-resisting channel so as to calibrate the signal provided by adownstream sensor.

[0076] The present invention optionally makes use of a multi-reservoirpressure controller coupled to a plurality of independently variablepressure modulators to effect movement of fluids within microfluidicnetworks, e.g., while providing monitoring of flow inducing pressures.By selectively controlling and changing the pressure applied to thereservoirs of a microfluidic device, hydrodynamic flow at very low flowrates can be accurately controlled within intersecting microfluidicchannels. Such pressure-induced flows can help to decrease (or entirelyavoid) any detrimental effects of the electrical fields associated withelectrokinetic transportation methods, such as sample bias, cellperforation, electroporation, and the like. Additionally, suchpressure-induced microfluidic flows can, through proper chip design,reduce flow variabilities as compared to electrokinetic techniquesthrough the use of pressure differentials (and/or channel resistancesthat are significantly greater than flow variations induced by secondaryeffects, such as inflow/outflow capillary force differentials within thereservoirs). Advantageously, the pressure-induced flows of the presentinvention can also be combined with electrokinetic and/or other fluidtransportation mechanisms, thereby providing compositepressure/electrokinetic microfluidic systems.

[0077] The techniques of the present invention will often make use ofdata regarding the network of channels within a microfluidic device.This network data can be calculated using a model of the microfluidicnetwork, measured by testing a microfluidic device, sensed using asensor, and/or the like. The network data will often be in the form ofhydrodynamic resistances along microfluidic channel segments connectingnodes, with the nodes often being intersections between channels, portsor reservoirs, connections between channel segments having differingcross-sectional dimensions and/or flow characteristics, and the like. Asused herein, the term “reservoir” encompasses ports for interfacing witha microfluidic network within a microfluidic body, including ports whichdo not have cross-sections that are much larger than the microfluidicchannel to enhance fluid capacity.

[0078] By selectively controlling the pressure at most or all of thereservoirs of a microfluidic system, very small flow rates can beinduced through selected channel segments. Such small pressure-inducedflows can be accurately controlled at flow rates which might bedifficult and/or impossible to control using alternative fluidtransportation mechanisms. Advantageously, the present invention canprovide flow rates of less than 0.1 nanoliters per second, the flowrates often being less than 1 nanoliters per second, and the pressureinduced flow rates typically being less than 10 nanoliters per secondwithin the microfluidic channel.

[0079] To accurately apply the pressures within the microfluidicnetwork, the invention can optionally make use of a pressuretransmission system having relatively large lumens coupling the pressuremodulators to the reservoirs of the microfluidic device, with thepressure transmission lumens ideally containing a compressible gas.Pressure is often transmitted through this relatively low resistancepressure transmission system to fluids disposed within the reservoirs ofthe microfluidic system via a gas/fluid interface within the reservoir.The resistance of the microfluidic channels to the fluid flows thereinis typically much greater than the resistance of the pressuretransmission lumens to the associated flow of compressible gas.Generally, the channel resistance is at least 10 times the transmissionsystem resistance, preferably being at least 100 times, and ideallybeing at least 1000 times the transmission system resistance of thecompressible gas used to induce the channel flows. In other words, aresponse time constant of the pressure transmission system willgenerally be lower than the time constant of the channel network,preferably being much lower, and ideally being at least one, two, orthree orders of magnitude lower. The head space of a fluid (for example,in the pressure modulator pump and/or in the port or reservoir) timesthe resistance of the fluid flow (for example, in the channels orlumens) generally define the response time constant.

[0080] It is often advantageous to enhance the resistance of themicrofluidic channels to provide the desired relative resistancefactors. The channels optionally have reduced cross-sectionaldimensions, pressure drop members (such as a small cross-sectionpressure orifice, a flow restricting substance or coating, or the like),and/or lengths of some, most, or even all of the microfluidic channelsegments are optionally increased by including serpentine segment paths.As the resistance of the pressure transmission system can be severalorders of magnitude less than the resistance of the channels, pressuredifferentials can be accurately transmitted from the pressure modulatorsto the reservoirs of the microfluidic device. Additionally, reducedtransmission system resistances can help to enhance the response of thepressure system, providing a faster response time constant.

[0081] Referring now to FIG. 1, a microfluidic system 10 includes amicrofluidic device 12 coupled to a bank of pressure modulators 14 by apressure transmission system 16. Pressure modulator bank 14 includes aplurality of pressure modulators 14 a, 14 b, . . . Modulator bank 14will generally include at least three independently, selectivelyvariable pressure modulators, typically having at least four modulators,and ideally having eight or more modulators. Each modulator is in fluidcommunication with a reservoir 18 of microfluidic device 12 via anassociated tube 20, the tube having a pressure transmission lumen with acompressible gas therein.

[0082] Modulator bank 14 generally provides independently selectablepressures to the lumens of tubing 20 under the direction of acontroller(s) 22. Feedback can be provided to controller 22 frompressure sensors 24, as will be described hereinbelow. Processor 22 willoften comprise a machine-readable code embodied by tangible media 26,with the machine-readable code comprising program instructions and/ordata for effecting the methods of the present invention. Processor 22can comprise a personal computer having at least an Intel Pentium® orPentium II® processor having a speed of at least 200 MHz, 300 MHz, ormore. Tangible media 26 can comprise one or more floppy disks, compactdisks, or “CDs,” magnetic recording tape, a read-only memory, a randomaccess memory, or the like. In some embodiments, the programminginstructions can be input into controller 22 via a disk drive or otherinput/output system such as an internet, intranet, modem reservoir, orthe like. Suitable programs can be written in a variety of programminglanguages, including the LabView™ language, as available from NationalInstruments of Austin, Tex. Controller 22 transmits drive signals tomodulator bank 14, ideally via an RS232/RS485 serial connection.

[0083] In addition to tubing 20, pressure transmission system 16includes a manifold 28. Manifold 28 releasably seals the lumen of eachtube 20 with an associated reservoir 18 of microfluidic device 12.Tubing 20 can comprise a relatively high-strength polymer such aspolyetheretherketone (PEEK), or a polytetrafluoroethylene (such as aTeflon™ material), or the like. The tubing typically has an innerdiameter in a range from about 0.01″ to about 0.05″, with a length fromabout 1 m to about 3 m. A “T” connector couples the pressure output fromeach pressure modulator to an associated pressure sensor 24.

[0084] Each modulator 14 a, 14 b . . . generally comprises a pump orother pressure source which pressurizes the compressible gas within thelumen of associated tubing 20. The modulators preferably comprisepositive displacement pumps, with the exemplary modulators comprising apiston which is selectively positioned within a surrounding cylinder byan actuator. Preferably, the actuators are adapted to allow accuratepositioning of the piston in response to drive signals from controller22, the exemplary actuators comprising stepper motors. The exemplarypiston/cylinder arrangement is similar to a syringe. Exemplary modulatorbanks can be provided by (or modified from components available through)a variety of commercial sources, including Kloehn of Las Vegas, Nev.,Cavaro of Sunnyvale, Calif., and the like.

[0085] Microfluidic device 12 is seen more clearly in FIG. 2.Microfluidic device 12 includes an array of reservoirs 18 a, 18 b, . . .coupled together by microscale channels defining a microfluidic network30. As used herein, the term “microscale” or “microfabricated” generallyrefers to structural elements or features of a device which have atleast one fabricated dimension in the range of from about 0.1 μm toabout 500 μm. Thus, a device referred to as being microfabricated ormicroscale will include at least one structural element or featurehaving such a dimension. When used to describe a fluidic element, suchas a passage, chamber or conduit, the terms “microscale”,“microfabricated” or “microfluidic” generally refer to one or more fluidpassages, chambers or conduits which have at least one internalcross-sectional dimension, e.g., depth, width, length, diameter, etc.,that is less than 500 μm, and typically between about 0.1 μm and about500 μm. In the devices of the present invention, the microscale channelsor chambers preferably have at least one cross-sectional dimensionbetween about 0.1 μm and 200 μm, more preferably between about 0.1 μmand 100 μm, and often between about 0.1 μm and 50 μm.

[0086] The microfluidic devices or systems of the present inventiontypically include at least one microscale channel, usually at least twointersecting microscale channel segments, and often, three or moreintersecting channel segments disposed within a single body structure.Channel intersections can exist in a number of formats, including crossintersections, “T” intersections, or any number of other structureswhereby two channels are in fluid communication.

[0087] The body structures of the devices which integrate variousmicrofluidic channels, chambers or other elements can be fabricated froma number of individual parts, which, when connected, form the integratedmicrofluidic devices described herein. For example, the body structurecan be fabricated from a number of separate capillary elements,microscale chambers, and the like, all of which are connected togetherto define an integrated body structure. Alternatively and in preferredaspects, the integrated body structure is fabricated from two or moresubstrate layers which are mated together to define a body structurehaving the channel and chamber networks of the devices within. Inparticular, a desired channel network is laid out upon a typicallyplanar surface of at least one of the two substrate layers as a seriesof grooves or indentations in that surface. A second substrate layer isoverlaid and bonded or fused to the first substrate layer, covering andsealing the grooves, to define the channels within the interior of thedevice. In order to provide fluid and/or control access to the channelsof the device, a series of reservoirs or reservoirs is typicallyprovided in at least one of the substrate layers, which reservoirs orreservoirs are in fluid communication with the various channels of thedevice.

[0088] A variety of different substrate materials can be used tofabricate the devices of the invention, including silica-basedsubstrates, i.e., glass, quartz, fused silica, silicon and the like,polymeric substrates, i.e., acrylics (e.g., polymethylmethacrylate)polycarbonate, polypropylene, polystyrene, and the like. Examples ofpreferred polymeric substrates are described in commonly owned publishedinternational patent application no. WO 98/46438 which is incorporatedherein by reference for all purposes. Silica-based substrates aregenerally amenable to microfabrication techniques that are well-known inthe art including, e.g., photolithographic techniques, wet chemicaletching, reactive ion etching (RIE) and the like. Fabrication ofpolymeric substrates is generally carried out using known polymerfabrication methods, e.g., injection molding, embossing, or the like. Inparticular, master molds or stamps are optionally created from solidsubstrates, such as glass, silicon, nickel electro forms, and the like,using well-known micro fabrication techniques. These techniques includephotolithography followed by wet chemical etching, LIGA methods, laserablation, thin film deposition technologies, chemical vapor deposition,and the like. These masters are then used to injection mold, cast oremboss the channel structures in the planar surface of the firstsubstrate surface. In particularly preferred aspects, the channel orchamber structures are embossed in the planar surface of the firstsubstrate. Methods of fabricating and bonding polymeric substrates aredescribed in commonly owned U.S. patent application Ser. No. 09/073,710,filed May 6, 1998, and incorporated herein by reference in its entiretyfor all purposes.

[0089] Further preferred aspects of the microfluidic devices of thepresent invention are more fully described in co-pending U.S. patentapplication Ser. No. 09/238,467, as filed on Jan. 28, 1999 (commonlyassigned with the present application), the full disclosure of which isincorporated herein by reference. These preferred aspects include, forexample, a reaction zone disposed within the overall body structure ofthe device, a reagent or other component of an “biochemical system”(generally referring to a chemical interaction that involves moleculesof the type generally found within living organisms), sensing systemsfor detecting and/or quantifying the results of a particular reaction(often by sensing an optical or other detectable signal of thereaction), and the like.

[0090] Referring once again to FIG. 2, reservoirs 18 will often bedefined by openings in an overlaying substrate layer. Reservoirs 18 arecoupled together by channels 32 of microfluidic network 30, with thechannels generally being defined by indentations in an underlying layerof the substrate, as was also described above.

[0091] Microfluidic channels 32 are in fluid communication with eachother at channel intersections 34 a, 34 b, . . . (generally referred toas intersections 34). To simplify analysis of microfluidic network 30,channels 32 can be analyzed as channel segments extending between nodesdefined at reservoirs 18 and/or channel intersections 34.

[0092] To provide enhanced control over movement of fluids withinmicrofluidic network 30 by reducing the effects of secondary hydrostaticforces (such as capillary forces within reservoirs 18), the resistanceof channels 32 to flow through the microfluidic network can be enhanced.These enhanced channel resistances can be provided by having a channellength greater than the normal separation between the nodes defining thechannel segment, such as by having serpentine areas 36 along the channelsegments. Alternatively, a cross-sectional dimension of the channel canbe decreased along at least a portion of the channel, or flow can beblocked by a flow restrictor such as a local orifice, a coating ormaterial disposed in the channel, or the like. In general, to takeadvantage of the full range of flow control provided by the pressuremodulators, microfluidic device 12 should be optimized for hydrodynamicflow. Flow control is generally enhanced by providing sufficient flowresistance between each reservoir 18 and the adjacent nodes so as toallow a sufficient variation in flow rate to be achieved within thevarious channel segments given the dynamic operating pressure range ofthe pressure modulators.

[0093] Pressure manifold 28 can be seen more clearly in FIGS. 3A and 3B.Manifold 28 has at least one device engaging surface 40 for engagingmicrofluidic device 12, with the engagement surface having an array ofpressure lumens 42 corresponding to reservoirs 18 of the device. Each ofpressure lumens 42 is in fluid communication with a fitting 44 forcoupling each reservoir with an associated pressure modulator via anassociated tube. Sealing body 46 helps maintain a seal between theassociated pressure modulator and reservoir, and manifold 28 isreleasably secured to device 12 by a securing mechanism 48, which hereincludes openings for threaded fasteners, or the like.

[0094] Manifold 28 can comprise a polymer, a metal such as 6061-T6aluminum, or a wide variety of alternative materials. Lumens 42 can havea dimension in a range from about 2 mm to about 3 mm. Fittings 44optionally comprise standard ¼-28 fittings. Sealing body 46 will oftencomprise an elastomer such as a natural or synthetic rubber.

[0095] The pressure transmission system (including manifold 28) willpreferably maintain a seal when transmitting pressures greater thanatmospheric pressure (positive gauge pressures) and less thanatmospheric pressure (negative gauge pressures or vacuum). The pressuretransmission system and modulator bank 14 will generally be capable ofapplying pressure differentials which are significantly higher thanhydrostatic and capillary pressures exerted by, for example, a buffer orother fluid in reservoirs 18, so as to avoid variability or noise in thepressure differential and resulting flow rates. As capillary pressureswithin reservoirs 18 are typically less than {fraction (1/10)} of a psi,often being less than {fraction (1/100)}th of a psi, the system willpreferably be capable of varying pressure at reservoirs 18 throughout arange of at least ½ psi, more often having a pressure range of at least1 psi, and most often having a pressure range of at least +/−1 psig (soas to provide a 2 psi pressure differential). Many systems are capableof applying at least about a 5 psi pressure differential, optionallyhaving pressure transmission capabilities so as to apply pressureanywhere throughout a range of at least about +/−5 psig.

[0096] A control system for selecting the pressures applied toreservoirs 18 is schematically illustrated in FIG. 4. Controller 22generally includes circuitry and/or programming which allows thecontroller to determine reservoir pressures which will provide a desiredflow within a channel of microfluidic network 30 (here schematicallyillustrated as microfluidic network controller 52) and also includescircuitry and/or programming to direct the modulators of modulator bank14 to provide the desired individual reservoir pressures (hereschematically illustrated as a plurality of pressure controllers 54.) Itshould be understood that network controller 52 and pressure controller54 can be integrated within a single hardware and/or software system,for example, running on a single processor board, or that a wide varietyof distributing process techniques might be employed. Similarly, whilepressure controllers 54 are schematically illustrated here as separatepressure controllers for each modulator, a single pressure controllercan be used with data sampling and/or multiplexing techniques.

[0097] In general, pressure controller 54 transmits drive signals to anactuator 56, and the actuator moves a piston of displacement pump orsyringe 58 in response to the drive signals. Movement of the pistonwithin pump 58 changes a pressure in pressure transmission system 20,and the change in pressure is sensed by pressure sensor 24. Pressuresensor 24 provides a feedback signal to the pressure controller 54, andthe pressure controller will optionally make use of the feedback signalso as to tailor the drive signals and accurately position the piston.

[0098] To enhance the time response of the pressure control system,pressure controller 54 can include pressure calibration data 60. Thecalibration data will generally indicate a correlation between drivesignals transmitted to actuator 56 and the pressure provided from thepressure modulator. Pressure calibration data 60 will preferably bedetermined by initially calibrating the pressure change system, ideallybefore initiation of testing using the microfluidic network.

[0099] Generation of calibration data 60 can be effected by transmittinga calibration drive signal to actuator 56 and sensing the pressureresponse using pressure sensor 24. The change of pressure from thiscalibration test can be stored in the program as calibration data 60.The calibration signal will typically cause a known displacement of thepiston within pump 58. Using this known displacement and the measuredchange in pressure, the overall pressure system response can becalculated for future drive signals using the ideal gas law, PV=nRT (inwhich P is pressure, V is the total compressible air volume, n is thenumber of moles of gas in the volume, R is the gas constant, and T isthe temperature). Calibration can be preformed for eachmodulator/pressure transmission systems/reservoir (so as to accommodatevarying reagent quantities within the reservoirs, and the like), or canbe preformed on a single reservoir pressurization system as an estimatefor calibration for all of the modulators of the system.

[0100] Once calibration data 60 has been generated, pressure controller54 can generate drive signals for actuator 56 quite quickly in responseto a desired pressure signal transmitted from network controller 52. Itshould be noted that these estimate will preferably accommodate thechanging overall volume of the compressible gas within the system, sothat the calculated change in pressure for a given displacement of thepiston within pump 58 at low pressures can be different than the samedisplacement of the piston at high pressures (i.e., thedisplacement/pressure correlation plot is not linear, but curves.).

[0101] In the exemplary embodiment, actuator 56 comprises a steppermotor coupled to a linear output mechanism. Pump 58 comprises a syringehaving a length of about 100 mm, and a diameter of about 20 mm. Overallresponse time for the system can depend on a variety of parameters,including dead volume, syringe size, and the like. Preferably, theresponse time will be less than about 1 sec/psi of pressure change,ideally being less than about 500 msecs/psi for a pressure change fromzero to 1 psi.

[0102] Network controller 52 generally calculates the desired pressurefrom each pressure modulator in response to a desired flow in one ormore of the channels of microfluidic network 30. Given a desired channelflow, network controller 52 derives these pressures using network data62, with the network data typically being supplied by either amathematical model of the microfluidic network 64 and/or a tester 66.Network data 62 will generally indicate a correlation between pressuredifferentials applied to reservoirs 18 and flows within the microfluidicchannels.

[0103] Network model 64 preferably comprises programming to helptranslate desired hydrodynamic flow rates into pressures to be appliedat reservoirs 18. An exemplary network model 64 generates a hydrodynamicmulti-level resistance network correlating to each microfluidic network30, as can be understood with reference to FIGS. 5A-5C.

[0104] Referring now to FIGS. 5A and 2, nodes can be defined at eachwell 18 and at each intersection 34. Hydrodynamic resistances of channelsegments coupling the nodes can be calculated from the chip design. Morespecifically, calculation of hydrodynamic resistances can be preformedusing hydrostatic pressure loss calculations based on the crosssectional dimensions of channels 32, the length of channel segmentsconnecting the nodes, the channel surface properties, the fluidproperties of the fluids included in the flows, and the like.

[0105] Analysis of the multi-level flow resistance network can beperformed using techniques often used for analysis of current inelectrical circuits, as can be understood with reference to FIGS. 5B-5C.Hydrodynamic resistances of the channel segments connecting reservoirs18 to adjacent nodes can be analyzed as the lowest level of amulti-level network. The channel segments adjoining these lowest levelsegments form the second level of hydrodynamic resistances of thenetwork. This level-by-level analysis continues until all channels ofmicrofluidic network 30 are included in the network model. The relativeflow rate of any channel in the microfluidic network can then beobtained once the flow rates from each of the reservoirs 18 in thelowest level have been calculated.

[0106] As described above, flow resistances are optionally calculatedbased upon hydrodynamic chip design alone. It is also possible tomeasure these resistances using, for example, electrical sensors,pressure drop sensors, or the like. In other words, resistances tohydrodynamic flow of the channels and channel segments can be measuredby, for example, measuring electrical resistance between reservoirs 18while a conductive fluid is disposed within the network. Regardless,once the channel resistances are known, the pressure drop in eachchannel segment in the network can be obtained by simply multiplying theflow rate of that channel with its associated channel resistance. Thepressure of each reservoir 18 can then be calculated by summing up allthe pressure drops along the network 30 starting at the top level of thenetwork.

[0107] Referring now to the exemplary program for calculating pressuresillustrated in FIGS. 5B and 5C, hydrodynamic flow rate Q is related toflow resistance R_(e) and pressure differential ΔP by the equation:

ΔP=Q·R _(e)

[0108] This relationship is quite similar to that used in electrokineticcalculations, in which current I and electrical resistance R are relatedto voltage V by the equation:

V=I·R

[0109] This simplifies the application of circuit analysis techniques tothe hydrodynamic analysis.

[0110] Determination of reservoir pressures so as to provide a desiredflow rate are preferably performed using pressure calculation program70, as illustrated in FIG. 5C. Desired flow rates are input in step 72from each reservoir 18. These flow rates can be input by the user, by anautomated test matrix generation program, or the like. Flow resistancesare obtained 74 as described above, and the input flow rate propagatesthrough the network to obtain flow rates for each branch 76. Thepressure drop of each branch is then determined using the networkresistance circuit 78. These pressure branches are then allowed topropagate through the network to obtain reservoir pressures 80 so as toeffect the desired flow.

[0111] Referring to FIG. 6, an alternative embodiment of a microfluidicsystem makes use of both electrokinetic transport and hydrodynamictransport mechanisms to move fluids (induce flow) within microfluidicchannels of the system. Electrokinetic transfer of fluids hassignificant advantages when electro osmosis and/or electrophoresis aredesired. Electrokinetic fluid transport is also both fast andconvenient, and modifications of the channel surfaces are possible toavoid and/or alleviate electrokinetic transport disadvantages. The plugprofiles of fluid plugs moved within a electrokinetic transport systemcan also be well-controlled and defined. As described in detailhereinbelow, controller 22 can be coupled to a sensor 154 fordetermining a viscosity of a sample fluid within the microfluidicnetwork. Alternatively, a separate processor can be provided forcalculating viscosities.

[0112] Electrokinetic/hydrodynamic system 90 also provides theadvantages of hydrodynamic transport described above. This hydrodynamictransport is quite reliable, and is independent of charges andelectrical surface properties of the channels. Hydrodynamic transport isparticularly well-suited for biocompounds which are sensitive toelectrical fields.

[0113] Electrokinetic/hydrodynamic microfluidic system 90 includes manyof the pressurization, microfluidic network and control componentsdescribed above. In this embodiment, manifold 92 includes fittings 44opening laterally from the manifold to provide sealed fluidcommunication from each pressure transmission tube 20 to an associatedreservoir 18 of the microfluidic device 12. Additionally, electrodes 94are coupled to each reservoir 18 via manifold 92. In the exemplaryembodiment, the electrodes comprise platinum surfaces which extend downfrom manifold 92 into electrical contact with fluids disposed withinreservoirs 18 when the manifold provides a sealing engagement betweenfittings 44 and the reservoirs. Coupling of the electrodes with thefittings 44 can be provided by using “T” connectors within the manifoldfor each well, and inserting a platinum electrode across and through the“T”. The appropriate (upper, in this example) connector branch of theT-connector can be sealed and the electrode affixed in place with asealing material such as epoxy.

[0114] By coupling electrodes 94 to computer 22, and by including withincomputer 22 an electrokinetic fluid transport controller capable ofinducing electro-osmosis and electrophoresis, the system of FIG. 6 iscapable of emulating pumps, valves, dispensers, reactors, separationsystems, and other laboratory fluid handling mechanisms, often withouthaving to resort to moving parts on microfluidic device 12.Electrokinetic transportation and control are described in, for example,U.S. Pat. No. 5,965,001, previously incorporated herein by reference.

[0115] One particular advantageous use of the pressure modulated flowcontrol can be understood with reference to FIGS. 7A and 7B. In manychemical analysis, it is desirable to vary the relative flow rates fromtwo reservoirs connected to a common node so as to vary a concentrationof a test solution, reagent, or the like, particularly for definingstandard curves of chemical reactions. As illustrated in FIG. 7A, it ispossible to vary the flows from two reservoirs electrokinetically, withthe relative fluid concentrations being indicated by the changes influorescence intensity over time. Unfortunately, control over therelative flow rates (and hence, the concentration) can be less thanideal due to variation in capillary forces within the reservoirs and thelike.

[0116] An alternative well-pair dilution plot in FIG. 7B can begenerated by varying concentrations using multi-pressure control. Thisplot illustrates the reduced noise and enhanced flow control provided bythe pressure control systems of the present invention. As generallydescribed above, hydrodynamic control can be enhanced by increasingresistance of the channel segments. In the exemplary microfluidic device12 illustrated in FIG. 2, channels 32 coupling wells 18 b, 18 c, 18 d,18 e, 18 f, and 18 g to the adjacent nodes have a resistance of 1.3×10¹¹g/cm⁴ s. Channel 32 coupling reservoir 18 a to the adjacent intersection34 has a resistance of 4.8×10¹⁰ g/cm⁴ S. Such a chip is well-suited foruse with flows having a pressure drop between reservoirs of about 2 psi,so as to provide a mixing time of about 6 seconds, and a reaction timeof about 20 seconds.

[0117]FIG. 7C is a plot of measured dilution vs. set dilution for adilution well-pair with a hydrodynamic flow system, showing the accuracyand controllability of these dilution methods. FIGS. 7D and 7E are plotsof the measured dilution near the upper and lower extremes,respectively, showing that a small amount of mixing at a channelintersection can occur when flow from a channel is at leastsubstantially halted. As can be understood with reference to thesefigures, some modification of the overall flow from one or more channelsat an intersection can be used to effect a desired dilution percentageadjacent a maximum and/or a minimum of the dilution range. For example,relative flow adjustments within 5% of a maximum or minimum desireddilution, and often within 2.5% of a desired maximum and/or minimum canbe employed. More specifically, to achieve a near 0% actual dilutionfrom a given channel at an intersection, fluid can flow into the channelat the intersection. Similarly, to achieve 100% measured dilution fromthe channel, more than 100% of the desired flow can be provided from thesupply channel into the intersection.

[0118] Characterization of an enzyme often involves determination ofmaximum reaction velocity and a Michaelis constant for each substrate.The enzymatic reaction of Alkaline Phosphatase on dFMUP (as illustratedin FIG. 8) was studied on a microfluidic device 12 optimized forpressure driven flow. FIG. 8A is a titration curve for differentconcentrations with and without substrate. A plot of backgroundcorrected signal vs. substrate concentration is shown in FIG. 8B, whilea Lineweaver-Burk plot for the Michaelis constant (Km) is provided inFIG. 8C. Results of a substrate titration assay for the reaction areshown in FIG. 8D.

[0119] Additional exemplary assay reactions, assay results, andmicrofluidic networks to provide those results are illustrated in FIGS.9A through 10B. More specifically, FIGS. 9A-C illustrate the reactionand assay results for a Protein Kinase A (PKA) assay performed atdifferent ATP concentrations. FIG. 10A illustrates a chip design havinga microfluidic network 130 of microfluidic channels 32 connectingreservoirs 18, in which the network is adapted for a mobility shiftassay. FIG. 10B are exemplary results of a mobility shift assay atdifferent concentrations of ATP as can be measured using the chip designof FIG. 10A.

[0120] Referring now to FIGS. 11A and 11B, an exemplary manifold or chipinterface structure 92′ is illustrated in more detail. Exemplarymanifold 92′ is adapted to provide both hydrodynamic coupling andelectrokinetic coupling between a microfluidic body and an associatedcontroller, as described above. Electrical conduit passages 140 forcoupling electrodes 94 to a system controller 22 (see FIG. 6) areillustrated in FIG. 11A. FIG. 11B illustrates manifold pressuretransmission lumens 142 which provide fluid communication betweenfittings 44 and a microfluidic body interface surface 144 withinmanifold 92. Manifold lumens 142 are illustrated in phantom.

[0121] Accurate control of the flow of fluids within a network ofmicrofluidic channels can be quite challenging within even a relativelysimple network of channels. More specifically, in many microfluidicapplications, a variety of different fluids (with differentcharacteristics) can be present in a single channel segment. Asdescribed above, where the hydro-resistance of each channel segment canbe obtained, it can be possible to simulate and calculate the flow offluids throughout the network for a given pressure configuration.Unfortunately, it can be quite difficult to accurately calculateviscosities (and, hence, resistances and flow rates) when severaldifferent buffers are used within a channel, often together with one ormore different test fluid samples.

[0122] Fortunately, a relatively simple flow sensor can be provided tomeasure an actual flow within a channel of a microfluidic network. Wherethe measured flow results from a known driving force (such as a knownpressure differential) can be determined, pressures to be applied at thefluid reservoirs so as to affect a desired flow condition can then becalculated.

[0123] Referring now to FIG. 12, a relatively simple viscometer 150makes use of a channel intersection 152 at a first location and adetector 154 at a second location to measure fluid flow characteristics.In general, a steady-state flow within a microfluidic channel 32 betweenintersection 152 and sensor 154 can be produced using a pressuredifferential between reservoirs 18, as described above. Intersection 152can impose a signal on the steady-state flow by applying a pressurepulse to one or more of the reservoirs 18, by applying an electrokineticpulse across intersection 152, or the like. The signal imposed atintersection 152 will often be in the form of a small flow perturbation,typically for a short duration. For example, where reservoir 18 dincludes a detectable dye, the flow perturbation or signal can comprisean increase or decrease in the dye concentration in the flow ofmicrofluidic channel 32 from intersection 152 toward detector 154.

[0124] Detector 154 is downstream from intersection 152, and can be usedto detect the arrival time of the signal, for example, as a peak or dipin the intensity of a fluorescent signal from the dye. Thus, the timedifference between imposition of the signal at intersection 152 andsensing of the signal flow at detector 154 can be readily measured.Calling this time differential Δt, and knowing the distance alongchannel 32 between intersection 152 and detector 154, Δd, from themicrofluidic network geometry, the flow rate Q can be calculated fromthe equation:

Q=A(Δd/Δt)

[0125] in which A is the cross-sectional area of the channel. Thismeasured flow rate of a steady-state flow for a given initial drivingforce greatly facilitates calculation of an appropriate pressureconfiguration to achieve a desired flow.

[0126] While the use of a pressure-controlled microfluidic flowgenerator (such as modulator bank 14) has significant advantages,alternative flow generators can be used. Volume-controlled microfluidicflow generators, for example, can also be used in a microfluidic systemto measure viscosity. A constant (or otherwise controlled) volumetricflow can be driven through a channel segment of a microfluidic networkusing known nanoliter scale syringe pumps. By measuring the pressuredifferential AP along the segment, and using the known volumetric flow Qgenerated by the pump, the flow resistance through the segment can becalculated. Viscosity can be determined from known channelcharacteristics and the flow resistance, and/or can be derived usingcalibration data generated by driving reference fluids having knownviscosities through the channel segment. Still further alternative flowgenerator means might be used, including capillary or spontaneous fluidinjection, electrically induced flow, and the like.

[0127] Where viscosity is to be determined by the system of FIG. 12,reservoirs 18 d and 18 e coupled to channel 32 by intersection 152 canindividually or in combination introduce fluid of known or unknownviscosity into the microfluidic channel at the intersection to provide aflow within the channel having an unknown total flow resistance. Withchannel 32 optionally containing only a trace amount of fluorescent dye(to inhibit any effect of the dye on the unknown overall viscosity), asubstantially constant pressure configuration at ports 18 can drive flowfrom intersection 152 toward detector 154. This steady-state flowcondition can be effected by a constant vacuum at reservoir 18 aadjacent detector 154, positive pressures applied at reservoirs 18 d, 18e adjacent intersection 152, or a combination of both. Regardless, thesteady-state flow with a constant pressure differential will result in avolumetric flow rate Q in channel 32 which is linearly proportional tothe pressure differential AP and inversely proportional to the fluidviscosity η as follows:

Q=KΔP/η

[0128] K is a proportionality constant which depends on the geometry ofthe channel network. K can be calculated from the channel geometry, orcan be determined through a calibration standard test, or the like.

[0129] When a pressure pulse is used to induce a flow perturbationgiving rise to a fluorescence intensity perturbation in viscometer 150,symmetric pressure pulsing can be applied at reservoirs 18 d and 18 esuch that the node pressure at 152 is unchanged to maintain a constantflow rate Q in channel 32 during the introduction of the dye pulse. Thissymmetric pulsing scheme mitigates the error in flow rate introduced bythe perturbation. It also allows the use of a relative long pulse toprovide a good signal-to-noise in the measured signal, even with asignificant amount of flow-induced dispersion. An alternative embodimentis to use symmetric pressure stopping from reservoirs 18 d and 18 e. Inthis case, the flow from 18 d steps down sharply in time as compensatingflow from 18 e steps up simultaneously. The total flow Q in channel 32is maintained, and the fluorescent intensity goes up and down as a stepfunction (rather than a pulse). The arrival time of the propagationfront of the dye (at 50% height of the final intensity value, forinstance) can be used to determine the time differential Δt.

[0130] A variety of alternative structures can be used to sense flowcharacteristics so as to apply a proper pressure configuration togenerate a desired flow. For example, a signal can be imposed on a flowwithin a microfluidic channel by photobleaching of a fluorescent dye,rather than imposing a flow perturbation at a intersection. Alternativeflow velocimetry approaches such as laser Dopler velocimetry, tracerparticle videography, and the like are also possible. Using suchtechniques, a simple straight channel connecting a fluid supplyreservoir and a waste fluid reservoir can suffice, with the fluid supplyreservoir containing a fluid comprising a photobleachable fluorescenttracer dye or appropriate tracer particles.

[0131] As can be understood with reference to the calculations of flowrate Q above, sensors can also be used to determine alternative flowcharacteristics within a microfluidic channel, including flow rate,viscosity, the proportionality constant for a segment or network (by useof fluids having known and/or uniform viscosities) and/or other flowcharacteristics. In fact, in addition to providing a tool to studyeffective viscosity of two or more mixed fluids (of optionally unequalviscosity) still further measurements are possible. Mixing of DMSO andan acquiesce buffer can yield a non-monotonic viscosity-compositionrelationship. By applying different levels of pressure differential ΔPand measuring the flow rate Q, viscometer 150 could be used to establisha relationship of the effective viscosity during mixing as a function ofmixing length. This information can be pertinent to chip design fortests which involve geometric dilution.

[0132] Where temperature dependency of viscosity is of interest, systemssuch as viscometer 150 can be coupled to a temperature control systemcomprising an external heater block in contact with the body definingthe microfluidic channel network, by using joule heating to selectivelycontrol the temperature of fluids within the channel network, or thelike. In a still further alternative, a structure similar to viscometer150 might be used to measure non-Newtonian viscosity. Non-Newtonianfluids have viscosities which are a function of the sheer rateexperienced by the fluid. One example of a non-Newtonian fluid is apolymer solution containing high molecular weight molecules. Amicrofluidic viscometer similar to viscometer 150 of FIG. 12 might havea channel geometry and/or channel network intersection structure and/orflow arranged so that the application of a pressure differential createsa range of sheer stresses so as to accurately measure such non-Newtonianviscosity.

[0133] Real-time flow control and viscosity measurements formicrofluidic systems based on transient pressure pulse techniques can befurther understood with reference to FIGS. 13A and 13B. A microfluidicnetwork structure 30 with a single branch channel coupling each node toa main channel 32′ is used. Each branch can be connected to a singlereservoir 18 for a different buffer, sample, enzyme, or like. In thesimplest embodiment, reservoir 18 e at the end of the microfluidicchannel network contains a dye solution to provide a detectable signal.

[0134] A steady flow can be directed toward reservoir 18 a by applyinginitial pressures on wells 18. A short pressure pulse can be applied towell 18 e and/or some or all of the other reservoirs of the microfluidicsystem. This pressure pulse will propagate substantially instantly toalter flow at some or all of the intersections 34 of network 30. Thisdisturbance of the flow at the node points can change the dilution ratiofrom one or more of the side branches. After the pressure pulse, steadystate flow is resumed.

[0135] As can be understood with reference to FIG. 13B, a time series ofsignals 160 a, 160 b, and 160 c occur at times T₁, T₂, and T₃,respectively. The flow rate from some or all of the side branches canthen be obtained from the difference of flow rates between successivenode points. Once the flow rates of the branches have been obtained, asthe pressures at reservoirs 18 are known, the resistances of the branchchannels can then be calculated. From the known channel geometry, theviscosity of the solution mixtures in main channel 32′ can be calculatedonce the viscosities of the solutions in the side branches connecting toreservoirs 18 b, 18 c, 18 d, and 18 e are known. This information canthen be fed back to the network model to derive the pressures for adesired flow rate from each reservoir.

[0136] In addition to providing the benefit of flow rate control, theflow perturbation technique also facilitates measuring viscosities ofsolution mixtures. For the network shown in FIG. 13A, the transit timeT₃ is a function of the viscosity of the mixture from 18 d and 18 e, T₂is a function of the viscosity of the mixture from 18 c, 18 d, and 18 e,and T₁ is a function of the viscosity of the mixture from 18 b, 18 c, 18d, and 18 e. Therefore, the microfluidic structure 30 can be used as aviscometer for multi-component solution mixtures. The fluid mixtures aredispensed and mixed microfluidically within the network. The compositionof the solution mixture in the main channel 32′ can easily be varied bychanging the pressures at the reservoirs thus changing the flow ratiofrom each side branch. Consequently, the complete behavior of viscosityas a function of composition can be mapped out relatively quickly withthis method.

[0137] The design of a viscometer for solution mixtures can be furtherrefined to take advantage of the symmetric pressure pulsing andsymmetric pressure stepping techniques of the detectable fluid. Anexample microfluidic structure is shown in FIG. 13C for a 3-fluidsystem. In this structure, each fluid reservoir has a paired wellcontaining the same fluid with trace fluorescent dye. Fluid 1 with andwithout dye can be loaded in reservoirs 18 g and 18 f, respectively,fluid 2 with and without dye in 18 e and 18 d, respectively, and fluid 3with and without dye can be loaded in reservoirs 18 c and 18 b,respectively. As a pressure gradient is applied to induce flow towardreservoir 18 a, symmetric pressure pulsing applied at each of thepairing reservoirs can be used to keep the node pressures at all channelintersections constant, thus maintaining constant flow rates and mixturecomposition in the main channel. The arrival times T₁, T₂, and T₃ of thepulses can be used to calculate mixture viscosities as discussed before.Alternatively, symmetric pressure stepping applied at each of thepairing reservoir yields the same advantages by analyzing the arrivaltimes of the dye fronts rather than dye pulses.

[0138] Referring now to FIGS. 14A and 14B, exemplary time signature dataindicates that pressure pulse signals can effectively be imposed on theflow within a microfluidic system, and can accurately and repeatedly besensed by a detector (such as an optical detector, or the like) formeasurement of flow characteristics.

[0139] Hydrodynamic, electrokinetic, and other fluid transport (flowinduction) mechanisms can be used in a variety of ways to providespecialized functions within a microfluidic system. For example, fluidmixtures such as biological fluid samples having particulates and/orcells in suspension within a liquid are often introduced intomicrofluidic systems. A particularly advantageous system and method forintroducing a large number of samples into a microfluidic system isdescribed in U.S. Pat. Nos. 5,779,868 and 5,942,443, the full disclosureof which is incorporated herein by reference. In that system, a vacuumcan be used to draw a sequential series of fluid samples from the wellsof a multi-well plate into a capillary tube in fluid communication withthe microfluidic system.

[0140] In the above-described system, it can be desirable to maintainfluids at a substantially stationary location within the microfluidicchannel, for example, during the time delay while a sample in a lastwell of a first multi-well plate is moved away from the capillary tubeand before a sample in a first well of a second multi-well plate is influid communication with the capillary tube. Maintaining the fluidswithin the microfluidic channel at a substantially fixed location canavoid introducing significant amounts of air into the microfluidicsystem, which might interfere with its operation. It can be desirable tomaintain fluid mixtures at a given location within a microfluidicnetwork for a wide variety of reasons.

[0141] Unfortunately, work in connection with the present invention hasfound that halting movement of some fluid mixtures within a microfluidicnetwork can have significant disadvantages. Specifically, cell-basedassays performed using a fluid mixture including cells suspended in aliquid are susceptible to sticking of the cells to the channel walls ifflow is completely halted. Similarly, other fluids can deteriorate ifflow within the channel is sufficiently low for a sufficient amount oftime.

[0142] To avoid deterioration of fluid mixtures, the present inventioncan provide a small amplitude oscillatory movement of a fluid mixture soas to maintain the fluid mixture within a microfluidic channel.Modulator bank 14 is capable of providing a small amplitude oscillatorypressure such that there is no significant inflow or outflow ofmaterials from the channel. This small amplitude oscillatory pressurewill preferably be sufficient to continuously move the fluid mixture(and, for example, the cells within the liquid) continuously back andforth. The oscillation frequency should be high enough such that theinstantaneous fluid mixture velocity is sufficiently high to avoiddeterioration of the mixture, while amplitude should be small enoughsuch that there is little or no unintended net transportation into orout of the channel from adjacent reservoirs, reservoirs and intersectingchannels. Once the desired delay in fluid mixture movement has beenprovided it will often be desirable to flow an intervening liquid suchas a buffer into the channel to help insure that unintended flows and/ormixtures at the channel ends have been flushed.

[0143] It should be noted that this small amplitude oscillatory motioncan optionally be provided using electrokinetic forces, such asproviding an alternating current, particularly if the alternatingcurrent is not harmful to cells or other components of the fluidmixture. It can be beneficial to insure that cells in the channel do notlyse when subjected to the alternating current if electrokinetic forcesare to be used to induce the oscillatory motion.

[0144] Referring now to FIG. 15, the systems and methods described abovecan optionally take advantage of a wide variety of pressure transientgenerators so as to initiate a flow perturbation. A multiple capillaryassembly 170 includes a microfluidic body or chip 172 mounted a polymerinterface housing 174. A plurality of capillaries 176 contain fluidintroduction channels. As explained in detail in U.S. Pat. No.6,149,787, the full disclosure of which is incorporated herein byreference, the capillary channels can be used to spontaneously injectfluids into the microfluidic network of chip 172 using capillary forcesbetween the injected fluid and the capillary channels. Such spontaneousinjection is sufficient to induce a pressure transient for measurementof hydrodynamic and/or electrokinetic flow. Such flow measurements allowthe derivation of information regarding the properties of the chip,microfluidic network, and/or fluids.

[0145] The use of multiple capillary assembly 170 is beneficial forparallel assays using a plurality of test samples, and the like.Referring now to FIG. 16, a simple chip 178 having a relativelystraightforward microfluidic network can be used to understand thederivation of flow and/or chip properties from spontaneous injection. Inmany embodiments, the open end of capillary 176 will be placed in afluid, typically by introducing the end of the capillary into amicrotiter plate (or any other structure supporting one or more fluidtest samples). This can be effected by moving the capillary 176 and chip178 relative to the microtiter plate, by moving the microtiter platerelative to the capillary or by moving both structures relative to eachother. Regardless, placing capillary 176 into a fluid results inspontaneous introduction of the fluid into the capillary channel. Byapplying a constant vacuum on at least one well of the microfluidicsystem, a steady flow can then be provided along a channel coupling thecapillary to the well.

[0146] If, for example, a steady-state flow is induced from capillary176, a substrate reservoir 180 a, and/or an enzyme reservoir 180 btoward a vacuum reservoir or waste well 180 c along a channel 182, aflow perturbation can be initiated at intersection 186 between thecapillary channel and the microfluidic network at the time the capillaryis withdrawn out from the well containing the introduced fluid. Thisflow perturbation can, for example, comprise a change in composition ofthe flow progressing along channel 182 toward vacuum reservoir 180 c.This change in composition can be sensed at a detection location 184 as,for example, a change in fluorescent intensity. Similar flowperturbations might be induced by applying other pressure transients atintersection 186, for example, when capillary 176 is introduced into thespontaneously injected fluid, or by applying a change in pressure usinga pressure modulation pump as described above, again changing thecomposition of the flow within channel 182. By monitoring the propertyof the composition at detection point 184, progress of the perturbationscan be detected. A time delay between initiation of the perturbation andtheir respective detections at the detection point, when combined with aknown length of channel 182, can be used to determine a speed of theflow within the channel. From this actual, real-time speed, a variety ofinformation regarding the fluid and/or network system can be determined.

[0147] Referring now to FIGS. 16A and 16B, each time capillary 176 isdipped into and are removed from a fluid well, a perturbation will begenerated at a capillary intersection 186 coupling the capillary channelwith the microfluidic network. Additionally, as the pressureperturbation will propagate throughout the microfluidic network, anotherflow perturbation can be simultaneously initiated at a secondintersection 186 a downstream of the sipper intersection 186. If weassume that fluid is flowing from reservoirs affixed to the microfluidicnetwork toward a vacuum reservoir 180 c, the pressure transient appliedby spontaneous injection at capillary 176 will alter the mixturesoccurring at each intersection.

[0148] Where the channel lengths can be designated, and Δd₁, Δd₂ a timedelay can be measured at detector 184 between initiation of the pressuretransient (at t=0) and sensing of a first flow perturbation as a signal188 a at detector 184. The first signal 188 a can be said to haveoccurred after a time delay of Δt₁, with this time being the timerequired for flow to propagate from the intersection immediatelyupstream of detector 184. A similar time delay Δt₂ will then be requiredfor the flow to propagate from the second upstream intersection (186 inthe simple network of FIG. 16A). Where the channel lengths betweenintersection are known, the various time delays can be used to determinethe various fluid speeds between intersections. Where the channelcross-sections are known, this information can be used to determinedcontributions from branch channels to the flow volume, and the like,regardless of whether the flows throughout the microfluidic system areinduced hydrodynamically, electrokinetically, electroosmotically, or thelike.

[0149] Referring now to FIG. 16C, capillary 176 can be dipped into andremoved from a variety of fluids in a sequential series. P indicatespressure, S₁ is a signal indicating a flow perturbation caused at afirst intersection by spontaneous injection into the capillary, andsignals S₂ indicates a flow perturbation signal generated at a secondintersection by the same spontaneous injection at the capillary. Aseries of pressure transients 190 will be generated by capillary 176when the capillary is, for example, dipped into and removed from a dye,followed by dipping of the capillary into a buffer solution, followeddipping of the capillary into a first test substance well, and the like.This sequence of spontaneous injection events at capillary 176 canresult in generation of a series of S₁ signals due to a series of flowperturbations at, for example, intersection 186 a. Simultaneously, aseries of second flow perturbation signals S₂ will also be generated atintersection 186, with detection of the second series following thefirst series by a time delay Δt₂ which is dependent on the speed offluid within the network channels. The total signal S_(t) measured atdetector 184 will be a combination of this offset series of signals withthe more immediate S₁ signals. Furthermore, the composition of theoverall flow arriving at the detector can vary significantly with thedifferent materials introduced by capillary 176. Regardless, by properlyidentifying the time delays between signals, flows between the nodes ofthe microfluidic system can be calculated.

[0150] Referring now to FIG. 16A, placing a detector 184 a downstream ofan electrode v₁ can facilitate measurements of electrically inducedflow, such as electroosmotic flows induced by a differential voltagebetween V₁ and V₂. As described above, pressure perturbations will beinitiated at the channel intersections, so that an initial signal can begenerated at the detector from the downstream electrode V₁, followed byanother signal generated at the upstream electrode V₂. Setting Δt₁, asthe time delay between these electrode intersections and Δt₂, as thetime delay for a subsequent signal generated by a reaction channel atintersection 186, and knowing the lengths of the channels Δd₁, Δd₂ wecan calculate the electroosmotic EO flow as follows: With voltagebetween the electrodes off, using only pressure to drive fluids withinthe network, we can determine velocities along the channels betweennodes caused by pressure v_(1P), v_(2P) from:$\frac{\Delta \quad t_{2}}{\Delta \quad d_{2}} = {{v_{2p}\quad {and}\quad \frac{\Delta \quad t_{1}}{\Delta \quad d_{1}}} = v_{1p}}$

[0151] While leaving the same pressure differential on, the voltagedifferential can then be turned on, allowing us to calculate theelectroosmotic flow velocity as follows:${\frac{\Delta \quad t_{2}^{1}}{\Delta \quad d_{2}} = {{v_{2p}\quad {and}\quad \frac{\Delta \quad t_{1}^{1}}{\Delta \quad d_{1}}} = {v_{1p} + v_{eo}}}};{{{which}\quad {gives}\quad {us}\quad v_{eo}} = \frac{{\Delta \quad t_{1}^{1}} - {\Delta \quad t_{1}}}{\Delta \quad d_{2}}}$

[0152] This electroosmotic velocity can then be used to calculateelectroosmotic mobility using the equation:${\mu_{eo} = \frac{v_{eo}}{E_{1}}},$

[0153] in which E₁ is the electric field strength between the first andsecond voltages V₁, V₂. FIG. 19 graphically illustrates data from adetector or sensor from which the time delays discussed above can betaken.

[0154] The multiple capillary assembly and simplified capillary networksof FIGS. 15, 16 and 16A are examples of microfluidic devices which mightbenefit from monitoring of pressure induced flow perturbations foranalysis and/or control of flows, quality control, and the like.Additional examples of microfluidic structures which can benefit fromthese techniques are illustrated in FIGS. 17A, 17B, 18A and 18B.

[0155] Referring now to FIGS. 17A and 17B, more complex microfluidicnetworks can include a plurality of capillary joints or intersections192 and substrate wells or reservoirs 194, enzyme wells 196, waste wells198, and the like. One or more detection or sensor windows or locations200 can be provided for monitoring of propagation of the flowperturbations. The microfluidic assembly and network of FIGS. 17A and17B can be useful for multi-capillary fluorogenic assays. Amulti-capillary basic mobility-shift microfluidic assembly and networkhaving similar structures is illustrated in FIGS. 18A and 18B. Thisstructure also includes a plurality of electrode wells 202 for applyingvoltages to the microfluidic network, as described above.

[0156] Accurate control of the flow perturbation pulse can bechallenging, particularly where the pulse comprises a step-functionchange in flow. To decrease noise in measurements, and to generallyenhance viscosity measurement accuracy and/or enhance measurement range,it can be beneficial to separate a flow having the perturbation(typically at an intersection within the network) into a plurality ofseparated flows. A first separated flow might advance along a firstseparated flow channel, while a second separated flow advances along asecond separated flow channel having dimensions (and thus a transittime) different than that of the first separated flow channel. As thechannel fabrication tolerances can be better controlled than the pulse,comparison of the transit times through the different channels can allowthe magnitude of a step-function pulse to be determined, and therebyallow more accurate viscosity calculations to be made.

[0157] Referring now to FIG. 20, a simple microfluidic network 220 fordetermining a viscosity of a fluid sample is shown schematically.Network 220 includes a sample fluid source 222 which supplies samplefluid to a first flow-resisting channel segment 224. A detectable fluidsource 226 is coupled to first segment 224 at an intersection 228 via asecond flow-resisting channel segment 230. A sensor or detector 154 isdisposed downstream of intersection 228, with the fluid from the sourceseventually flowing to a waste well 232 or the like.

[0158] In network 220 shown in FIG. 20, the fluid sources and waste wellcan comprise simple ports or reservoirs as described above. By usingthis simple arrangement with three wells and a single intersection,viscosities of sample fluids can be determined without mixing the samplefluid with a dye or other detectable substance in such a manner as toalter the viscosity to be measured. Specifically, the channels ofnetwork 220 can be completely (often filled using capillary action) witha reference fluid having a known viscosity. The reference fluid used toinitially fill the channels of the network can be free from dyes orother detectable substances. The fluid sample can be introduced intosample fluid source 222, and reference fluid having a detectablesubstance such as a dye can be introduced into detectable fluid source226. A flow of the fluids within the network can be generated towardwaste well 222, for example, by applying a steady vacuum at the wastewell.

[0159] Referring now to FIGS. 20, 21A and 21B, where the reference anddetectable fluid have similar viscosities, the dilution of the dye-freereference fluid initially used to fill the channels of network 220 canfirst be determined by the relative hydrodynamic resistances of thefirst and second channel segments 224, 230 (the parallel sections of thenetwork circuit), as described below. This can result in a substantiallysteady signal at the detector 154 after initiation of the flow, (withthe baseline signal shown in the figures being generated prior to anyflow of the detectable fluid past the detector). Optionally, thisinitial calibration flow can proceed with reference fluid disposedwithin sample fluid source 222, and/or with the detectable fluidpre-filling second segment 230. Regardless, this initial flow can bedominated by the flow resistance of the reference fluid within the firstchannel segment and the detectable fluid within the second channelsegment so as to produce a steady reference signal, as schematicallyshown in FIGS. 21A and 21B.

[0160] As a significant portion of the sample fluid is drawn into thefirst channel segment 224, the ratio between the resistance the firstchannel segment to the reference fluid and the resistance of the firstchannel segment to the flow of the sample fluid therein is altered. Thisresults in more or less dye (or other detectable fluid) entering theflow at intersection 228. For example, if the fluid sample is moreviscous than the reference fluid initially disposed within the firstchannel segment 224, the signal will increase at detector 154. Thischange in signal at the detector results from the change in thepercentage of detectable fluid in the combined flow at the detector. Asillustrated in FIG. 21A, in our example of a sample fluid having aviscosity higher than that of reference fluid, the signal at detector154 will increase due to this increased percentage or ratio ofdetectable fluid at the detector. Similarly, in the case of a samplefluid which is less viscous than the reference fluid, overall resistanceof the first channel segment 224 to the flow there through willdecrease, and less detectable fluid will be present in the flow atdetector 154, resulting in a reduction in the signal generated bydetector 154, as illustrated in FIG. 21B. Hence, the relative viscosityof the sample fluid can be determined by comparison to the flowcharacteristics of the reference fluid. The signals illustrated forFIGS. 21A and 21B can be any appropriate detector signal units, and areschematically shown as Reference Fluorescent Units (“RFU”), as might beappropriate for the use of a fluorescent dye in the detectable fluid.Suitable reference fluids might include, for example, aqueous solutionsof ethylene glycol having known concentrations, thereby providing knownviscosities from about 1 to about 15 cp. For many embodiments, suitablereference fluids can comprise (or be composed of) water with one or moredye (optionally being diFMU or Fluorescein), a buffer (optionally beinga well-known buffer such as HEPES, TRIS, or CAPS at an appropriate pHfor the associated dye) and a dye, or the like. Suitable referencefluids can also comprise (or be composed of) a National Institute ofStandards and Technology (NIST) (or other standard regulating body)certified viscosity standard, which are generally oils having a knownviscosity at a given temperatures. Such viscosity standards arecommercially available, and are used for calibration of traditionalviscometers.

[0161] It should be recognized that the schematic graphs shown in FIGS.21A and 21B are a simplification. Changes in the signal can beginimmediately and/or gradually as the sample fluid enters theflow-resisting segment, and the signal need not be linear.

[0162] From the above, it can be understood that the viscosity of thesample can be analyzed as a function of the slope of the fluorescentsignal, with positive slopes representing samples that are more viscousthan the reference, and negative slopes representing samples that areless viscous than the reference fluid. The relationship betweenviscosity and the slope of the fluorescence signal will typically not belinear. Nonetheless, the relationship is generally reproducible, so thatthe relationship can be mapped by fitting a curve (such as a polynomial,an exponential expression, or the like) to the viscosity/signal slopedata. The relationship can generally be trendfitted/overlayed oncalibration or other measurement data to allow the viscosity to bedetermined as a function of the signal slope. Still further approachesmight be used, including generation of a look-up table and the like.

[0163] Still further related methods can be used to determine theviscosity from a sensor signal. For example, as the sample fluid anddetectable fluid from the sources fill the channel network and achievesteady state flow, the signal from detector 154 should likewise leveloff. Hence, the viscosity of the sample fluid can be determined inresponse to the change in slope of the signal prior to achievingsteady-state flow, and/or by using the eventual steady-state signal.

[0164] Determination of the viscosity of the sample fluid might beperformed using the relative volumetric flow rates through theflow-resisting channel segments as related to fluid viscosity in theequations given above. However, actual resistances to flows within amicrofluidic network can be quite sensitive to channel dimensions.Processing and fabrication capabilities can, in turn, limit how tightlythe dimensions or tolerances of a particular chop are constrained. As aresult, accuracy of viscosity measurement will often benefitsignificantly from at least one calibration measurement.

[0165] Viscosity measurement accuracy will typically be enhanced bytaking a plurality of calibration measurements for a specific network,the measurements generally being taken with fluids at a plurality ofdifferent known viscosities. As the signal will often vary with changesin viscosity with a non-linear relationship, three or more calibrationmeasurements can be taken with each chip, typically from three to sevenmeasurements. From a plurality of signal slopes or other measurementstaken with reference fluids having known viscosities, a calibrationcurve can be established, and an equation determined fitting thefluid-flow and signal response for a particular microfluidic chip andits network. Measurements taken with the sample fluid will then providea fluorescence signal (or other) response which can be mapped onto thecalibration curve to determine the sample viscosities. Using thisreference fluid-based approach the involved calculations can comprise(or even be limited to) derivation of the calibration curve frommeasurements taken with reference fluids, together with mapping ofmeasurements taken with sample fluids onto the calibration curve todetermine sample viscosity.

[0166] A number of network design variations around the generalmicrofluidic viscometer concept described above can be implemented. Insome embodiments, alternative (and in some cases slightly morecomplicated) viscometer channel networks can provide a larger possibledynamic range, enhanced accuracy, greater ease of use, or the like. Onealternate viscometry network channel 240 is illustrated in FIG. 22.Network 240 includes many of the components described above regardingnetwork 220, with the flow path coupling the sample fluid source 222 tointersection 228 here comprising a first segment portion 242, a secondsegment portion 244, and a third segment portion 246. The hydrodynamicresistance of the channel segment portion 244 has been locally enhanced,so that the majority of the flow-resistance of the sample fluid betweenthe sample fluid source 222 and the intersection 228 is imposed alongthe second channel segment portion 244. This can be effected by alteringthe depth of the channel along this segment portion, altering the widthof the channel segment along this portion, by placing a flow occludingstructure within the channel along this segment portion, or the like.Optionally, the bulk of the resistance can be localized within arelatively short channel length, for example, by taking advantage of thestrong dependence of the hydrodynamic resistance on the channel depth,and by locally decreasing that channel depth.

[0167] As can be understood with reference to FIGS. 23A and 23B, locallyenhancing hydrodyamic resistance R2 along second channel segment portion244 can help to provide signals from detector 154 which do not changesignificantly until the flow front has entered and/or filled channelsegment 244. After a steeply ramped change in signal, little additionalchange in the detector signal will occur, assuming that R2 is muchlarger than R3 or R1. Hence, the signal response to the network 240would substantially be in a form of a stair step, in which the magnitudeof the step is proportional to (or otherwise a function of) theviscosity, with samples having greater viscosity than a reference fluidgenerating an increased signal as illustrated in FIG. 23A, while samplefluids having a lower viscosity than the reference fluid generating astair-step down signal as illustrated in FIG. 23B.

[0168] It can be advantageous to enhance the dynamic range of amicrofluidic viscosity measurement system as can be understood withreference to FIGS. 24 and 25. In network 250 of FIG. 24, a series offlow resisting channel segments 224 a, 224 b, 224 c are aligned inseries. Detectable fluid sources 226 a, 226 b, 226 c are coupled to theseries flow path by associated channel segments 230 a, 230 b, 230 c atassociated intersections 228 a, 228 b, 228 c. This series ofhydrodynamic resisters of the sample fluid flow can result in a seriesof signal plateaus as the sample passes each sequential intersection. Byanalyzing the relative plateau levels, it will be possible to determineviscosity of the sample fluid, potentially over several orders ofmagnitude. While three detectable fluid branch channels are shown, twoor more can also be used.

[0169] In one exemplary embodiment, detectable fluid source 226 a andits associated channel of components can provide for detection ofviscosities in a range from about 1 to about 10 cp. Similarly, a seconddetectable fluid source 226 b can be used for detection of viscositiesin a range from about 10-100 cp, while a third detectable fluid sourcemy provide viscosities in a range from about 100-1000 cp. The variationin sensing range associated with the different detectable fluid sources,is enhanced by the differences in percentage change of flow at thedifferent intersections. Differing side resistances and/or differingdetectable fluids having differing signatures (as discussed belowregarding FIG. 25), differing detectable fluid viscosities from thediffering detectable fluid sources, and the like can also be employed.

[0170] Another wide dynamic range network 260 is illustrated in FIG. 25.In this embodiment, a first detectable fluid source 262 includes a firstdetectable fluid (for example, a dye having a first color signal) and asecond detectable fluid source 224, having a second independentlydetectable fluid (for example, having a dye with a second color signal).Detectable fluid sources 262, 264 are coupled to the firstflow-resisting channel segment 224 by a second channel segment 266, anda third channel segment 268, respectively. Segment 266 has asignificantly lower hydrodynamic resistance to flow of the firstdetectable fluid than segment 268 has to flow of the second detectablefluid therethrough. When a vacuum is drawn at waste well 232, a mixtureratio between the sample fluid from sample fluid source 222, and thefirst detectable fluid can indicated viscosities in a relatively lowrange (for example, 1-10 cp), while mixing of the sample fluid with thesecond detectable fluid can indicate viscosities throughout a higherviscosity rating (for example, through out a range of 10-100 cp). Hence,by simply using (for example) dyes having different color together withside channel segments having varying fluidic resistances, themicrofluidic viscometry range can be significantly enhanced.

[0171] In another embodiment, the viscometer 220 can be used to measurethe viscosity of a sample fluid that is immisciable with the detectablefluid. The flow rate of the sample fluid through the capillary isinfluenced by its viscosity as well as the surface tension propertiesbetween the two fluids. The pressure differential across the immisciablefluid interface is determined at least in part by the interfacialtension, the contact angle with the capillary wall, and the radius ofthe capillary. It is therefore possible to measure interfacialproperties between immisciable liquids in addition to sample viscosityby monitoring the changes in the fluorescence intensity signal as afunction of time, which is indicative of the sample flow rate asdiscussed earlier.

[0172] Referring back to FIGS. 20 and 6, independent pressure controlprovided by modulators 14 can also be used to determine viscosity usingfeedback from sensor 154. A quantity of a sample fluid having an unknownviscosity can be introduced to reservoir 222, through a capillary 176,or the like. Reference fluid can optionally be introduced before andafter the sample fluid to produce a “plug” of sample fluid in channel224, and the viscosity of the sample fluid within the network willaffect relative flows at intersections upstream and/or downstream of theplug. A feedback control loop from sensor 154 to pressure modulators 14allows pressures from port 226 to be adjusted until a desired signalresponse is achieved. For example, when the plug is first introducedinto the main channel, the signal from a dye flowing from reservoir 226will increase in slope if the plug has a high viscosity. Pressure atreservoir 226 might be decreased until the signal response is flatagain. By knowing how much the pressure was changed and the geometry ofthe chip, one can determine how much the sample fluid has changed theflow resistance in the network, and can thereby determine viscosity ofthe sample fluid.

[0173] Referring now to FIG. 26, the use of microfluidic viscometry in ahigh-throughput system can be understood. High-throughput microfluidicsystems and methods are more fully described in PCT Publication No. WO00/10015, the full disclosure of which is incorporated herein byreference. In simple terms, a microfluidic body 270, having channelwalls defining the channels of a microfluidic network is coupled to acapillary 272 for entering sample fluids 274 a, 274 b, and 274 c . . .in the channel network. The sample fluids can be disposed in the wellsof a microtiter plate 276, with the plate (and/or microfluidic body 270)being moved robotically so as to sequentially introduce the samplefluids through capillary 272. High-throughput systems can also bemultiplexed so as to simultaneously determine a plurality ofviscosities, optionally with a plurality of capillaries extending from asingle body.

[0174] To account for variation between capillaries (differences indiameter, coupling of the capillary to the microfluidic network, and thelike) it can be advantageous to calibrate the viscosity measurements byintroducing a reference fluid from a well of microtitre plate 276. Insome embodiments, the bulk of the fluidic resistance can be incorporatedin the microfluidic body (rather than the capillary) to reduce thesensitivity of a high-throughput system and method to capillaryvariability, capillary junction connection variability, and the like.

[0175] A variety of alternative microfluidic networks might be used tomeasure viscosity and/or other fluid characteristics. Using the conceptsdescribed above, viscosity might be determined by comparativelymeasuring flow resistance through a segment of such a network in amanner analogous to the well-known comparative methods used forelectrical resistance measurements in the Wheatstone bridge circuit.Such a microfluidic network can be particularly well-suited forviscosity measurements of fluid mixtures, and can also facilitateviscosity measurements throughout a range of dilutions. Other fluidcharacteristics might also be measure in such a network, includingelectrical conductivity or the like.

[0176] As can be understood from the network design tools describedabove, a channel simulation program can be helpful in optimizingpotential microfluidic viscometer body designs. Calibrations data for aspecific microfluidic network can enhance measurements accuracy, as isalso explained above. FIGS. 27-33 show measurements and simulated dataof viscosities as measured with the microfluidic techniques describedherein. Specifically, FIG. 27 shows measurement data generated using amicrofluidic chip (NS71, fabrication by Caliper Technologies Corp. ofMountain View, Calif.) by “sipping” (introducing via a capillary 272)sequential 20-second flows of sample fluids having varying viscosities.The sample fluids for these measurements were solutions of ethyleneglycol varying from 0 to 100%, providing viscosities varying from about1 to about 15 cp. FIG. 28 shows corresponding data generated by acomputer model or simulation of this microfluidic system.

[0177]FIGS. 29 and 30 show measurement data and simulation data,respectively, for a single 20-second sip a 10% ethylene glycol solution.The figure shows spontaneous injection of the sample within thecapillary due to capillary forces. Viscosity measurements can be basedat least in part on the rate of change of the fluorescence (in thiscase, the increase) when sipping the sample fluid. Measurement data fora rate of change or slope for a 20-second sip of 40% ethylene glycol isshown in FIG. 31, while corresponding simulation data is shown in FIG.32. The initial change in fluorescence can be the maximum if any errorsresulting from spontaneous injection are neglected. These graphs wereagain generated using the NS71 chip.

[0178] Information from three measurements for the various titrations ofethylene gylcol are summarized in FIG. 33. As the viscosities of thesamples are known, these measurements allow a calibration curve to bedetermined for the chip. Measurement data from the calibrated chip canbe referenced to the calibration curve to determine viscosity. Forexample, if a sample material results in a fluorescence slope of 0.06,the curve indicates that the sample material has a viscosity of about4.5 cp.

[0179] Three curves are shown in FIG. 33, with each curve beinggenerated for an associated experiment. The reproducibility of theexperiments is indicated by the correspondence of the curves.Sensitivity is higher at the lower viscosities as there is a moresignificant separation in slopes for a given change in viscosity.Accurate viscosity measurement range can be enhanced by the structuresand methods described above. The simulation results deviate somewhatfrom the measurement data, which can (at least in part) result fromactual channel dimensions, channel surfaces, capillary dimensions,capillary/chip joints, and the like, which vary from the computer model.

[0180] Techniques are provided in the invention for performing accurateviscosity measurements in which a single perturbation in a detectablesignal (e.g., fluorescence intensity) can be used as a signal signatureto determine viscosity of a fluid. In overview, a fluid comprising asignal moiety (e.g., a fluorophore) is directed into two or morechannels with defined geometries and different hydrodynamic resistance.Based on the relative flow rates of the fluid in the two channels,viscosity can be measured or calculated. Because the method relies onchannel geometry rather than applied forces, and because one channeleffectively acts to calibrate the other, the method is highlyreproducible and reduces errors or “noise” in viscosity measurements.

[0181] For example, as illustrated in FIGS. 34A-F, one can design thegeometry of first and second microfluidic channel segments (channel Aand channel B in FIG. 34) such that they have significantly differentflow transit times, allowing a single signal perturbation (e.g.,introduction of a detectable marker pulse into the channels) to be splitinto two signals, e.g., at a channel intersection (node), with thedifference in flow time characterizing the viscosity of the fluid. Ineffect, channel B becomes an internal calibration channel for channel A,to provide signal repeatability of <1% variation over repeated use of achip with precise control over channel geometry (such as channel lengthand channel cross section shape). For example, in a T-Junction pulseviscometer (a perpendicular junction is not a requirement of theembodiment, i.e., a T-junction” as used here generically, refers to “T”shapes, “Y” shapes and other similar channel geometries), thisreproducibility eliminates errors that arise from the generation of asignal perturbation (e.g., flow of detectable components). This alsoreduces restrictions on the duration of the pressure or electrokineticpulse used to produce the signal that is detected.

[0182]FIG. 34, panel A illustrates an example microfluidic channelgeometry that can be used to perform precise viscosity measurements whenusing this pulsed-injection/perturbation method. For example, in theillustrated embodiment, the same fluid (e.g., a 1 centipoise fluid ) isloaded throughout the chip, but a reservoir containing the fluid is alsospiked with a small amount of fluorescein such that a perturbation influorescence signal (resulting from flow of label from the reservoirpast a detector) can be produced by inducing a pulse of fluid from thereservoir. The graph to the right of panel A shows the (e.g.,fluorescence) readout at the detector. FIG. 34, panel B illustrates theresults of a pulse induced via pressure or electrokinetic action (ΔV orΔP), as per the T-junction viscometer. For example, undervacuum/hydrodynamic pulse induction, the fluid plug comprising thesignal reagent is induced to flow through the chip, e.g., towardsvacuum. As shown in FIG. 34, panel C, the pulse is split into two. Inthis example, because the transit time from channel B is slower thanchannel A (due to the channel geometry and resultant hydrodynamicresistance), the pulse in channel B arrives later at the detector. InFIG. 34, panel D, the two signal perturbations arrive at the detector,separated, in this illustration, by 3 seconds from each other, resultingin the fluorescence signature shown to the right in the plot. Asillustrated in FIG. 34, panel E, for a fluid of 10 cp (10× higher thanthe previous example) the transit times through the channels areincreased proportionally (10× in this example). The transit times forthe signal perturbations (e.g., plugs of dye or other detectablematerials) are also increased proportionately. As illustrated in FIG.34., Panel F, the relationship between viscosity and transit timedifference is linear. It will be apparent to those skilled in the artthat the absolute viscosity of a sample fluid can be determined, e.g.,by comparing the transit times of one or more reference standards ofknown viscosity to sample fluid transit times in the same chip.

[0183] In another embodiment, the viscosity of a sample fluid can bedetermined by evaluation of flow inducing pressures required to set anon-mixed interface at a particular location in a detection channel.This embodiment is based on controlling the location of a non-mixedinterface between the two fluids, one typically a reference standard,after they are brought together, e.g., at a T-intersection. Detailsregarding analytical solutions to Navier-Stokes equations that describeflow of two immiscible incompressible liquids of different constantabsolute viscosity in a rectangular channel are found in Gambos andForster (1998) “An Optical Micro-fluidic Viscometer” MEMS 66:187-191 andin U.S. Pat. No. 6,134,950, “Method for Determining Concentration of aLaminar Sample Stream”, to Forster, et al.

[0184] In this embodiment, viscosity can be measured by determining theforce of flow induction pressures at fluid wells (or other reservoirs)that is required to set the fluid interface at a selected region (e.g.,the centerline) of a low-resistance detection channel, downstream of theT intersection. This embodiment can makes use of multi-reservoirpressure control and, e.g., two-layer etch technology (the latter allowsthe resistance of the channel following the intersection to be madenegligible as compared to the channels leading directly to the referencefluid well and the well containing the fluid of unknown viscosity). Flowcan be measured by any detection method, including optical detection(e.g., where one or more of the fluid streams are fluorescent),conductivity detection, or the like.

[0185] As illustrated in FIG. 35, a reference fluid (typically havingknown viscosity properties) and a fluid comprising properties to bedetermined are induced to flow simultaneously into a T intersection fromchannels A and B and into detector channel C. The reference fluid ofknown viscosity (μ₁) having a flow rate Q₁ at pressure P₁, and thesample fluid of unknown viscosity having a flow rate of Q₂ at pressureP₂. Either fluid or both fluids can contain detectable marker asappropriate for the detection method. The pressures (P₁ and P₂) areadjusted until the interface of the two fluids is positioned at aselected location in the channel (e.g., the centerline). For thecondition illustrated in FIG. 35:

Q ₂ /Q ₁ =ΔP ₂ /ΔP ₁(μ₁/μ₂)=f(μ₂/μ₁).

ΔP ₂ /ΔP ₁=(f(μ₂/μ₁))/(μ₂/μ₁), where (μ₂/μ₁)=g(ΔP ₂ /ΔP ₁).

[0186] In general, ΔP=P−P_(n)≈P if the pressure drop between P_(n) andP_(w) is negligible. Thus, viscosity can be calculated if the appliedpressures to bring the fluid interface to a selected location in channelC are known. For example, as graphically illustrated in FIG. 36, if massflows are equal, and the interface is located at the centerline ofrelatively wide channel C (a wide aspect ratio of the channel crosssection) the ratio of the pressures required to obtain the centerlineinterface is approximately equal to the ratios of the fluid viscosities(P₂/P₁≈μ₂/μ₁). Where channel C is relatively deep, the viscosity of asample fluid can be determined from, e.g., the known reference fluidviscosity and known flow induction pressures with reference toexperimentally prepared calibration curves.

[0187] In another aspect, the invention provides a chip-based viscometerthat allows for the determination of mass-percent composition (thepercentage of mass in a fluid mixture that is attributable to any onefluid contributing to the mixture) and kinematic viscosity (dynamicviscosity corrected for mass) of a flowing mixture of two or more fluidstreams. The chip utilizes available reference fluid density andviscosity information; the dilution and mixture viscosity are measuredsimultaneously. The embodiment relies on multi-reservoir pressurecontrol and imaging of a marker front (e.g., fluoroscein, conductivesalt solutions, and the like) to set a zero-flow condition on a sidechannel connecting to the main channel that contains the mixed fluids.Once the zero-flow condition is set, the pressure at the intersection ofthe side channel and the main channel is equal to the pressure appliedto the side-channel well. Thus, a multi-reservoir controller and opticalimaging are used to measure the pressure drop in the main channel; themixture composition and viscosity is determined from the measuredpressure drop and the pressures applied to the fluid wells.

[0188] The invention provides a chip-based viscometer that allows forthe determination of mass-percent composition (the percentage of mass ina fluid mixture attributable to any one contributing fluid stream) andkinematic viscosity (dynamic viscosity corrected for mass) of a flowingmixture of two or more fluid streams. The chip utilizes availableinformation of reference fluid densities and viscosities; the dilutionand mixture viscosity are measured simultaneously. The embodiment relieson multi-reservoir pressure control and imaging of a marker front (e.g.,fluoroscein, conductive salt solutions, and the like) to set a zero-flowcondition on a side channel connecting to the main channel that containsthe mixed fluids. Once the zero-flow condition is set, the pressure atthe intersection of the side channel and the main channel is equal tothe pressure applied to the side-channel well. Thus, a multi-reservoircontroller and optical imaging are used to measure the pressure drop inthe main channel; the mixture composition and viscosity is determinedfrom the measured pressure drop and the pressures applied to the fluidwells.

[0189] For example, to simultaneously measure mass-percent compositionand kinematic viscosity, a microfluidic chip, as shown schematically inFIG. 37A, can be employed. Fluid well 290 can contain, e.g., a firstfluid of known kinematic viscosity and second fluid well 291 can containa second fluid of known kinematic viscosity. The wells can beindependently pressurized (e.g., using a multi-reservoir pressurecontrol manifold) to provide desired fluid flows into mixing node 293,past side channel node 294, and along main channel section 295 to wastewell 232. Side channel well 296, containing, e.g., a detectable marker,such as a dye reagent or salt solution, can be pressurized to create amarker front along side channel 297 that can be detected by detector298. The pressure in side channel well 296 can be adjusted to a levelequivalent to the pressure at side channel node 294 as confirmed bydetection of a stationary marker front (the pressure at the node thenmeasurable as the pressure in the side channel well). A stable dynamicflow is thus created, e.g., wherein the mass-percent composition foreach fluid and the kinematic viscosity of the fluid mixture can becalculated.

[0190] Calculations for the system described above can be based on themomentum equation for a fully developed flow of an incompressibleliquid, given by:

ΔP=mvrL, or

v=ΔP/mrL

[0191] where kinematic viscosity (v) of a fluid is equal to the pressuredifferential (ΔP) of the fluid flowing in a channel segment divided bythe product of the fluid mass flow rate (m), the channel resistanceshape factor (r), and the channel length (L). In well characterizedsystems, many of these parameters are well known, e.g., from theoreticalcalculations and experimentation. In the system of FIG. 37A, forexample, the kinematic viscosity (v) of a mixed fluid in main channelsection 295 can be calculated after determining the pressuredifferential between side channel node 294 (P₂₉₄, measured at well 296under zero-flow side channel conditions) and waste well 232 (P₂₃₂,typically constant, and often 0 psig). Once P₂₉₄ is known, P₂₉₃ can becalculated, e.g., from the known resistance ratios of channel segment295 and channel segment 300:

P ₂₉₃=(P ₂₉₄ −P ₂₃₂)(r ₃₀₀ L ₃₀₀ +r ₂₉₅ L ₂₉₅)/(r ₂₉₅ L ₂₉₅)+P₂₃₂

[0192] Once P₂₉₃ is calculated, the mass flow rate from well 291 (m₂₉₁)can be calculated, e.g., using the calculated P₂₉₃ and the known (orpreviously determined) properties r₂₉₉, L₂₉₉, and v₂₉₁:

[0193]m ₂₉₁=(P ₂₉₁ −P ₂₉₃)/(v ₂₉₁ ×r ₂₉₉ ×L ₂₉₉)

[0194] The mass flow rate from well 290 can be calculated in a similarmanner and the total mass flow rate in channel 300 (which is the same asthat in channel 295 under the zero-flow condition) is calculated as:

m ₃₀₀ =m ₂₉₅ =m ₂₉₀ +m ₂₉₁

[0195] The kinematic viscosity can then be calculated as the pressuredifferential between side-channel node 294 and well 232 divided by theproduct of the total mass flow (m₂₉₅) and the resistance along mainchannel segment 295 (r₂₉₅×L₂₉₅). That is:

v ₂₉₅=(P ₂₉₄ −P ₂₃₂)/(m ₂₉₅ ×r ₂₉₅ ×L ₂₉₅)

[0196] The composition of a fluid mixture (percentage of each comprisingfluid, m %) can be calculated from quantities that were determined orcalculated in the manner described above:

m%₂₉₉ =m ₂₉₉ / m ₂₉₅

[0197] By varying the pressures on wells 290 and 291, and thendetermining the mixture composition and viscosity, a curve can begenerated that describes kinematic viscosity of the fluid mixture versusmass-percent composition (e.g. v₂₉₅ versus m %₂₉₉).

[0198] The pressure of fluid at any location along a channel can bemeasured, e.g., at a side channel fluid well where the side channel flowhas been set to zero-flow. The zero-flow condition can also bedetermined using, for example, an electrical conductivity/resistancemeasurement. Such a measurement can be performed using a system, e.g.,similar to that shown schematically in FIG. 37b. In this case, wells 3and 4 can be filled, e.g., with a fluid having an electricalconductivity much higher (or lower) than the fluids in wells 1 or 2. Todetermine the zero-flow condition, the same pressure can be applied atwells 3 and 4 and this pressure can be, e.g., adjusted while theelectrical resistance of the side channel is simultaneously measured(e.g. by sending a DC or AC current from well 3 to well 4 and measuringthe resulting voltage differential). Given the conductivity mismatch ofthe fluids in wells 1 and 2 with those of wells 3 and 4, flow into orout of the side channel will cause a change in electrical resistancebetween wells 3 and 4. When the flow in the side channel is stopped, theelectrical resistance of the side channel will remain constant,indicating a zero-flow condition in the side channel. The pressureapplied to wells 3 and 4, in this case is, e.g., an indirect measure offluid pressures (Pn₂) at the intersection of the side channel with themain channel.

[0199] In further embodiments of viscometers of the invention, a chipcan include, e.g., paired channel geometry to provide an internalstandard for channel etch depth and temperature compensation. Such achip, as shown in FIG. 38, can be used to determine the viscosity of anunknown sample relative to a reference fluid of known viscosity. Forexample, reference fluid well 280 can contain a reference fluid of knownviscosity and sample fluid source well 222 can contain a sample fluidwith a viscosity to be determined. A diluent fluid, e.g., containing adetectable marker, such as a dye, can be contained in diluent fluid well281. Each of the three wells can be equally pressurized, e.g., with amulti-reservoir pressure manifold, to induce fluids to flow into wastewell 232. After fluid flows have stabilized, the difference in amountsof diluent fluid (detectable marker) at detectors 282 a and 282 b can bedetermined. If the sample fluid viscosity is higher than the referencefluid viscosity, more diluent fluid containing detectable marker willflow into reference channel 283. The sample fluid viscosity can bedetermined by, e.g., calculation, or by reference to an experimentallygenerated calibration curve of measured dye ratios for the reference andknown sample calibration reagents.

[0200] Sample fluid viscosity can optionally be compared to referencefluid viscosity using the paired channel configuration of FIG. 38, e.g.,by measurement of a time difference output value for a detectable markerpulse traveling in the paired channels (typically having equivalentvolume and resistance). For example, the three fluid wells can beequally pressurized to establish a stable flow. A pressure pulse can beexerted on diluent well 281 to inject a detectable marker pulse in theflow of channels 283 and 284. If the sample fluid is less viscous thanthe reference fluid, the pulse will arrive, e.g., at detector 282 bbefore detector 282 a. The sample fluid viscosity can be determined by,e.g., calculation, or by reference to an experimentally generatedcalibration curve of measured pulse travel time differences between thereference and sample calibration reagents of known viscosity. For thisviscosity measurement technique, a single detector, e.g., at wastechannel 285, can be optionally used to detect the reference and samplepulse, as can be appreciated by those skilled in the art.

[0201] Sample fluid viscosity can optionally be compared to referencefluid viscosity using the paired channel configuration of FIG. 38, e.g.,by measurement of a pressure differential output value necessary toobtain equivalent flow rates in the paired channels. For example, thethree fluid wells can be equally pressurized to establish stable fluidflows. A pressure pulse can be exerted on diluent well 281 to inject adetectable marker pulse in the flow of channels 283 and 284. If thesample fluid is more viscous than the reference fluid, the pulse willarrive, e.g., at detector 282 a before detector 282 b. The pressureinducing flow from sample fluid well 222 can then be, e.g., increased,and a second marker pulse introduced into the fluid flows. If the pulsearrives, e.g., at detector 282 a again before the pulse at detector 282b, the pressure inducing flow from sample fluid well 222 can beincreased further before another pulse timing measurement. Afterrepeated pulse timing and pressure adjustment cycles, e.g., a pressuredifferential between reference well 280 and sample well 222 can bedetermined which provides equivalent travel times for pulses in channels283 and 284. The sample fluid viscosity can be determined by, e.g.,calculation, or by reference to an experimentally generated calibrationcurve of measured pressure differences required to obtain equivalentpulse travel times between the reference and a set of sample calibrationreagents of known viscosity. Simple programs can be written, e.g., inMatlab to measure the arrival time of pulses in the chip channel and toiteratively adjust the chip well pressures.

[0202] In an additional embodiment, the invention provides methods formeasuring viscoelasticity (relaxation time) of polymers such asbiopolymers, ordered or disordered micellar solutions and/or surfactantsin a microfluidic system. The method operates by measuring the transientresponse of a test fluid that is following through a micro channel underthe action of an applied sinusoidal pressure gradient of frequency ω.

[0203] For example, a simple T-junction chip is used to illustrate thisapproach in FIG. 39. Although a perpendicular intersection alignment ispreferred in this invention, other geometries can be employed. Adetectable marker labeled Newtonian fluid (such as fluorescein in a lowconcentration buffer solution) is loaded in well #371, a polymericsample fluid is loaded in well #378, and both fluids are mixed at theT-junction and induced to co-flow towards waste well #374. The dilutionratio of the fluids depends upon the applied pressure of wells 371 and378 (pressure at well 374 can, e.g., =0 psi). If the flow inducingpressure at well #378 is made to oscillate as: p=p_(o) cosωt, thedilution ratio of the polymeric sample fluid can be shown to oscillateas Q₃₇₈/Q₃₇₁˜ap_(o) {(1+bλω²)cos ωt−(λ−c)sin ωt} where a, b and c aredimensional constants, Q₃₇₈/Q₃₇₁ represents the ratio of sample fluidand Newtonian fluid mass flows, and λ is the relaxation time of the testpolymeric fluid. Hence, if the detector is located near the T-junction,it will register a sinusoidally varying signal (oscillations in theamount of detectable marker) with an additional phase shift associatedwith the time constant λ. This signal can then be compared with theinput pressure oscillation to determine the phase shift. The phase shiftvalue can be used in a calculation of the test polymeric materialviscoelasticity. For a simple Newtonian fluid, the pressure and detectorsignal will be out of phase (λ=0 s). Various time scales in the flow ofthe polymeric solutions can be probed using a spectrum of pressurefrequencies.

[0204] There are a number of applications for this method. For example,this method can determine the viscoelasticity (relaxation times) ofpolymeric solutions such as long chain biopolymers, lipids, entangledwormy micellar solutions (see, FIG. 40 for a schematic diagram ofmicellar system phase changes that can affect viscoelasticity), liquidcrystals, vesicles and spherical micelles. These systems tend to changetheir viscoelastic behavior with concentration, temperature and ionicstrength (if an amphiphilic molecule is charged). Hence, the effects ofthe parameters can be easily probed using a transient microchip. Forexample, a solution of 15 mM cetylpyridinium salicylate in the presenceof 11 mM sodium salicylate has a relaxation time of ˜1 s. Uponincreasing the ionic strength by 14%, the relaxation time of theresulting solution increases by two orders of magnitude (˜100 s) (Rehageand Hoffman, Journal of Physical Chemistry, 92, 4712, 1988). Thestructure and state of aggregation of concentrated ionic surfactants inelectrolytic solutions can also be inferred from these measurements.

[0205] It should be understood that the various embodiments of methodsand structures described herein will often be combined. For example,dilution and/or fluid combination structures and methods can be combinedin a microfluidic network with viscometer structures and methods,allowing fluid mixtures to be characterized as a function of theircomposition. Mixtures will often include more than two fluids, and theability to controllably mix three or more, five or more, and/or ten ormore fluids in a controlled manner within the network allows complexstudies to be performed quickly and with small fluid volumes. Measuredfluid flow, measured flow times, and/or viscometry information derivedfrom the network will, as noted above, often by used by the fluid flowgenerator control system to control the flow of fluids within thenetwork via a feedback control loop. Similarly, computer simulations ofnetworks can be combined with flow times and other flow measurement datato enhance feedback control of flows within the network.

[0206] While the viscosity measurement structures described herein havegenerally, for purposes of illustration, made use of pressuredifferential, similar microfluidic systems might make use of other flowgenerating systems within a microfluidic environment for determiningviscosities of small quantities of sample fluids. In general, while theexemplary embodiments have been described in some detail, by way ofexample and for clarity of example, a variety of modifications, changes,and adaptation will be obvious to those of skill in the art. Hence, thescope of the present invention is limited solely by the appended claims.

[0207] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. A microfluidic viscometer system comprising: amicrofluidic channel network including a first flow-resisting channelsegment; a sensor coupled to the first segment of the network fordetermining a viscosity of a sample fluid therein.
 2. The microfluidicsystem of claim 1, further comprising a body having channel wallsdefining the network, the network including a plurality of channels withintersections therebetween.
 3. The microfluidic system of claim 2,further comprising a flow generator coupled to the network so as toinduce a flow of the sample fluid within the first segment.
 4. Themicrofluidic system of claim 3, wherein a first intersection is incommunication with the first segment, the sensor coupled to the networkat a sensor location disposed downstream of the first segment, thesensor sensing a change in the flow which propagates from the firstintersection to the sensor location so as to determine the viscosity ofthe sample fluid.
 5. The microfluidic system of claim 4, wherein thechange in flow comprises a pulse of a detectable fluid introduced at thefirst intersection, the first intersection being upstream of the firstsegment, the system determining the viscosity of the sample fluid inresponse to a steady state propagation of the flow, with the detectablefluid pulse, from the first intersection, through the first segment, tothe sensor location.
 6. The microfluidic system of claim 4, wherein thechange in flow comprises a step change in flow of a detectable fluid. 7.The microfluidic system of claim 4, wherein the first segment isdisposed upstream of the first intersection, wherein the flow defines aratio between a quantity of the sample fluid in the flow and a quantityof a detectable fluid in the flow, the detectable fluid being detectableby the sensor and traversing a second flow resisting channel segmentbetween a detectable fluid source and the intersection, wherein thechange in flow comprises a change in the ratio, and further comprising aprocessor coupled to the sensor, the processor determining the viscosityof the sample fluid from the change in the ratio and from the viscosityof the reference fluid.
 8. The microfluidic system of claim 1, whereinthe sample fluid for which the viscosity is determined is substantiallyfree of a substance detectable to the sensor.
 9. The microfluidic systemof claim 1, further comprising a sample fluid source including aplurality of sample fluids and a sample fluid introduction channel, thesample fluids sequentially transferable along the fluid introductionchannel to the flow resisting channel so as to sequentially determineviscosities of the sample fluids.
 10. The microfluidic system of claim1, wherein the first channel segment comprises an upstream end, adownstream end, and a first hydrodynamic resistance, the system furthercomprising a second flow-resisting channel segment comprising a secondhydrodynamic resistance and intersecting the first segment at theupstream end and at the downstream end.
 11. The microfluidic system ofclaim 10, further comprising an instruction set to compare transit timesof one or more fluid through the first segment and the second segmentfor determination of a fluid viscosity.
 12. The microfluidic system ofclaim 1, further comprising: a side channel well in fluid contact withthe first channel segment through a side channel at a side channelintersection; a detector coupled to the side channel for detecting flowin the side channel; and, a pressure sensor coupled to the side channelwell to measure a side channel well pressure for determining a pressureat the side channel intersection.
 13. The microfluidic system of claim1, further comprising: a second flow-resisting channel in fluid contactwith the first channel through a diluent channel; a diluent fluid,comprising a detectable marker, within the diluent channel; a sensorcoupled to the second channel, whereby the marker is detected fordetermining a viscosity of one or more fluids in the system.
 14. Amicrofluidic viscometer system comprising: a sample fluid channelcomprising a first pressure detector; a reference fluid channelcomprising a second pressure detector; a detector channel in fluidcontact with the sample channel and the reference channel at aT-intersection; and, a detector coupled to the detector channel fordetecting a fluid interface at a selected location within the detectorchannel.
 15. The microfluidic viscometer of claim 14, wherein theselected location is a centerline of the detector channel.
 16. Amicrofluidic viscometer system for determination of viscoelasticity, thesystem comprising: a sample fluid channel in fluid contact with an flowgenerator for induction of an oscillating fluid flow; a reference fluidchannel in fluid contact with the sample fluid channel at anintersection; a detector channel in fluid contact with the samplechannel and the reference channel at the intersection; a detectorcoupled to the detector channel and configured to detect oscillations inan amount of detectable marker a fluid; and, an instruction setconfigured to determine a phase shift between a fluid oscillation and adetectable marker oscillation for determination of a fluidviscoelasticity.
 17. A method for determining a viscosity of a samplefluid, the method comprising: altering a flow of a flow-restrictingmicrofluidic channel segment; and determining the viscosity of thesample fluid by monitoring the altered flow.
 18. The method of claim 17,further comprising: monitoring a first flow of reference fluid throughthe flow-resisting channel segment, the reference fluid having a knownviscosity; monitoring a second flow through the flow-resisting channelsegment, the second flow comprising the sample fluid; and wherein theviscosity of the sample fluid is determined by comparing the first andsecond flows, and based in part on the known viscosity of the referencefluid.
 19. The method of claim 18, wherein the first and second flowsare monitored by a sensor disposed downstream of the flow-resistingchannel segment with an intersection disposed therebetween, and whereinthe flows are monitored by sensing a ratio of the sample fluid to adetectable fluid, the detectable fluid being combined with the samplefluid at the intersection.
 20. The method of claim 18, furthercomprising sequentially transferring a plurality of sample fluids to theflow-resisting channel segment and determining the viscosities of thesample fluids in a high throughput manner.
 21. The method of claim 18, aplurality of microfluidic channels and the flow-resisting channeldefining a microfluidic network, further comprising mixing first andsecond fluids in the network, the mixed fluids defining the samplefluid, and varying a composition of the sample fluid by changingrelative quantities of the first and second fluids in the mixing step soas to determine the viscosity of the mixed fluids as a function of thecomposition.
 22. A microfluidic system comprising: a microfluidicchannel network including a first flow-resisting channel segment; asensor coupled to the network for sensing flows through the firstsegment; and a processor coupled to the sensor, the processor deriving aviscosity of a sample fluid by comparing first and second flows throughthe first segment.
 23. The microfluidic system of claim 22, furthercomprising a reference fluid disposed within the network, the first flowcomprising the reference fluid, the second flow comprising the samplefluid.
 24. The microfluidic system of claim 23, wherein the second flowwithin the first segment is substantially composed of the sample fluid.25. The microfluidic system of claim 23, wherein the processorcalculates the viscosity of the sample fluid from the source based atleast in part on a viscosity of the reference fluid.
 26. Themicrofluidic system of claim 22, further comprising a secondflow-resisting channel segment coupled to the first segment at a firstintersection, wherein a first detectable fluid is disposed within thesecond segment, the first segment being disposed downstream of the firstintersection, wherein the sensor monitors the flow through the firstsegment by sensing a quantity of the first detectable fluid added to theflow at least in part at the first intersection.
 27. The microfluidicsystem of claim 26, further comprising at least one additionalflow-resisting channel segment coupled to the first segment by anassociated at least one additional intersection, the intersections beingseparated by intersection separating flow-resisting channel segments,wherein the sensor monitors the flow by sensing a quantity of the firstdetectable fluid added to the flow at the intersections.
 28. Themicrofluidic system of claim 26, further comprising a thirdflow-resisting channel segment coupled to the first segment, wherein thesensor further monitors the flow through the first segment by sensing aquantity of a second detectable fluid added to the flow through thethird segment, the second and third segments having differingresistances to flows therein, the first and second detectable fluidsbeing independently detectable by the sensor.
 29. The microfluidicsystem of claim 26, wherein the first segment comprises a channel regionhaving a locally enhanced resistance to flows therein.
 30. Themicrofluidic system of claim 26, wherein the first flow comprises areference fluid having a known viscosity, and wherein the second flow atthe sensor comprises a combination of the sample fluid and thedetectable fluid, the combination defining a ratio, the processoridentifying the ratio from a signal produced by the sensor.
 31. Themicrofluidic system of claim 22, wherein the processor derives theviscosity of the sample fluid by determining a rate of change of asignal generated by the sensor.
 32. The microfluidic system of claim 22,wherein the processor derives the viscosity of the sample fluid bydetermining a magnitude of a change of a signal generated by the sensor.33. The microfluidic system of claim 22, wherein the processor candetermine the sample fluid viscosity throughout at least a range ofabout there orders of magnitude of cp units.
 34. The microfluidic systemof claim 33, wherein the processor can determine the sample fluidviscosity throughout at least a range from about 1 cp to about 100 cp.35. The microfluidic system of claim 34, wherein the processor candetermine the sample fluid viscosity throughout at least a range fromabout 1 cp to about 1000 cp.
 36. The microfluidic system of claim 22,further comprising a sample fluid source including a plurality of samplefluids and a sample fluid introduction channel, the sample fluidssequentially transferable along the fluid introduction channel to theflow resisting channel so as to sequentially determine viscosities ofthe sample fluids.
 37. The microfluidic system of claim 36, wherein thesample introduction channel comprises a capillary extending from amicrofluidic body, the microfluidic body having channel walls definingthe network, the capillary extendable sequentially into the samplefluids.
 38. The microfluidic system of claim 37, wherein the capillaryis extendable into a reference fluid having a known viscosity, the firstflow comprising the reference fluid.
 39. The microfluidic system ofclaim 37, wherein the capillary has significantly less resistance to theflow than the first segment.
 40. The microfluidic system of claim 23,further comprising a differential pressure source coupled to thenetwork, the pressure source applying a pressure differential urging thesample fluid through the first segment.
 41. The microfluidic system of40, wherein the pressure source applies a vacuum downstream of the firstsegment, the vacuum drawing the sample fluid through the first segmentand a first intersection, the vacuum also drawing a detectable fluidthrough a second flow-resisting channel segment and the firstintersection, the processor determining the viscosity in part from asignal of the sensor determining a quantity of the detectable fluid. 42.The microfluidic system of claim 22, further comprising a fluid volumesource coupled to the network, the volume source introducing a volume offluid at a known rate into the first segment.
 43. A microfluidic systemcomprising: a microfluidic body having a network of microfluidicchannels; a fluid flow generator coupled to the network and inducing aflow therein; a sensor coupled to the network, the sensor transmitting asignal indicating a time of the flow; and a processor coupling thesensor to the generator and effecting feedback control of the flow inresponse to the time signal.
 44. The microfluidic system of claim 43,wherein the processor determines a viscosity of a fluid in the flow andmodifies a driving force applied to the network by the generator inresponse to the viscosity.
 45. A microfluidic system comprising: firstand second immisciable fluids; a microfluidic body having a microfluidicnetwork, the first and second fluids being combined within the network;and a sensor coupled to the network so as to define a viscometer formeasuring interfacial properties of the combined fluids.
 46. A methodfor determining a viscosity of a sample fluid, the method comprising:flowing the sample fluid through two or more microfluidic channelsegments of different hydrodynamic resistance; and, monitoring adifference in transit times of sample fluid in the channels; whereby theviscosity of the sample fluid is determined based on the difference intransit times.
 47. The method of claim 46, further comprising: comparingthe difference in transit times of the sample fluid to a difference intransit times for a reference fluid of known viscosity; whereby theviscosity of the sample fluid is determined based on the known viscosityof the reference fluid.
 48. A method for determining a viscosity of asample fluid, the method comprising: flowing the sample fluid in a firstmicrofluidic channel; flowing a reference fluid in a second microfluidicchannel which converges with the first channel forming a T-intersection,wherein the sample fluid and reference fluid form an interface flowingfrom the T-intersection into a third micro channel; and, adjusting alocation of the interface in the third channel by modifying one or morepressures, which one or more pressures control flow of the fluids;wherein the viscosity of the sample fluid is determined based in part ona difference in pressures between the first and second channels, orbased on a known viscosity of the reference fluid.
 49. A method fordetermining a viscosity or a mass-percent composition of a fluidmixture, the method comprising: flowing one or more fluids into amicrofluidic channel; setting a side channel pressure on a side channelintersecting the microfluidic channel at a first location to provide azero-flow condition in the side channel, thereby determining anintersection pressure; and, determining a pressure difference betweenthe intersection pressure and a microfluidic channel pressure at asecond location; thereby providing parameters for determination of theviscosity or mass-percent composition of the fluid mixture.
 50. Themethod of claim 49, wherein setting the side channel pressure comprisesdetecting a marker in a side channel fluid, and adjusting the sidechannel pressure to provide unchanging detection of the marker, therebysetting the side channel pressure to provide a zero-flow condition. 51.The method of claim 50, wherein detecting a marker comprises measuringconductivity, fluorescence, light absorption, or refraction.
 52. Amethod for determining a viscosity of a sample fluid, the methodcomprising: flowing a reference fluid of known viscosity in a firstchannel intersected by a diluent channel in fluid contact with a diluentfluid comprising a detectable marker; flowing a sample fluid in a secondchannel intersected by the diluent channel; flowing the diluent fluidinto the first or second channels; and, detecting the diluent fluid;whereby the viscosity of the sample fluid can be determined based on oneor more outcome values.
 53. The method of claim 52, wherein the outcomevalues comprise a difference in amounts of detectable marker detected inthe first channel and the second channel.
 54. The method of claim 52,further comprising: injecting a pulse of detectable marker from thediluent channel into the first and second channels; and, measuringtravel times of the pulse in the first and second channels; wherein theoutcome values comprise one or more travel time difference for thepulses in the first and second channels.
 55. The method of claim 52,further comprising: monitoring one or more pressures inducing flow inthe first and second channels; and, injecting a pulse of detectablemarker from the diluent channel into the first or second channels;wherein the outcome values comprise one or more pressure differencebetween the first and second channels.
 56. A method of determining aviscoelasticity of a sample fluid, wherein the method comprises: flowinga reference fluid in a first microfluidic channel; inducing anoscillating flow of a sample fluid in a second microfluidic channelwhich intersects with the first channel to form a third microfluidicchannel comprising a detector, wherein the sample fluid or referencefluid comprises a detectable marker; monitoring oscillations in theamount of detectable marker in the third channel; comparing the samplefluid flow oscillation to the detectable marker oscillation, therebydetermining a phase shift; and, determining the viscoelasticity of thesample fluid based in part on the phase shift.